SINGLE EXPRESSION VECTOR FOR GENERATION OF A VIRUS WITH A SEGMENTED GENOME

Abstract
The present invention encompasses an expression vector that is capable of generating a virus from a segmented genome. In particular, a single expression vector may be utilized to produce influenza virus in cultured cells. The expression vector can be delivered in a purified DNA form or by a suitably designed bacterial carrier to cells in culture or to animals. This invention increases the virus generation efficiency, which benefits vaccine development. The bacterial carrier harboring such a plasmid encoding an attenuated virus may be used as a vaccine against corresponding viral disease.
Description
FIELD OF THE INVENTION

The invention encompasses an expression vector and a bacterial carrier. The expression vector is capable of generating a virus after being delivered into host cells. The bacterial carrier of the invention may be utilized to deliver the expression vector into host cells. The virus produced in the host cells from the expression vector may be either attenuated or not attenuated.


BACKGROUND OF THE INVENTION

Influenza virus has caused three recorded pandemics. The 1918 influenza pandemic, also known as Spanish influenza, caused at least 675,000 deaths in the U.S. alone and up to 50 million deaths worldwide (1, 34). The 1957 influenza pandemic caused at least 70,000 deaths in U.S. and 1-2 million deaths worldwide (2, WHO). The 1968 influenza pandemic caused about 34,000 deaths in U.S. and 700,000 deaths worldwide (2, WHO). Since 2003, there were 411 human cases and 256 deaths of avian influenza from 15 countries (WHO). The estimated mortality is more than 60%, making the highly pathogenic H5N1 avian influenza virus a potential candidate for the next influenza pandemic. The economic consequences of such a pandemic due to morbidity and health care delivery would be staggering.


The annual economic burden of influenza epidemics is also enormous. During a typical year in the United States, 30,000 to 50,000 persons die as a result of influenza virus infection, and the global death toll is about 20 to 30 times higher than the toll in this country (26). Based on the 2003 US population, annual influenza epidemics result in an average of 610,660 life-years lost, 3.1 million hospitalized days, and 31.4 million outpatient visits with the total direct medical costs averaging up to $10.4 billion annually. Projected lost earnings due to illness and loss of life amounted to $16.3 billion annually. The total economic burden of annual influenza epidemics using projected statistical life values amounted to $87.1 billion (20). The aforementioned socio-economic factors make influenza one of the critical infectious agents and hence a vaccine to prevent the resulting pandemics is highly warranted.


The three-recorded pandemics and most yearly global outbreaks of influenza are caused by influenza A virus (3, 13, 31, 32, 35). The virus belongs to the family Orthomyxoviridae, and contains a segmented negative-strand RNA genome. Influenza viral RNAs (vRNAs) associate with influenza RNA polymerase complex (PBI, PB2, PA), and nucleoprotein (NP) to make up a set of ribonucleoproteins (RNPs) (14, 21, 25). RNPs are both critical and essential constituents that mediate transcription or replication of vRNA. RNP can be reconstituted in vitro by incubating purified influenza polymerase and nucleoprotein with vRNA transcribed from template DNA (17). The reconstituted RNP has catalytic properties very similar to those of native viral RNP complexes. In the presence of influenza helper virus the recombinant RNP can be amplified and packaged into virus particles in a eukaryotic host cell, a process commonly known as RNP transfection (17) that also enables site-directed mutagenesis of any single component of the influenza virus genome (8). However, the need to select recombinant virus from the mixture of helper viruses and low viral yield demand more sophisticated approaches for the construction of recombinant influenza virus for the production of vaccines that need to be modified annually.


Effort to construct recombinant influenza virus using modern genetic tools for potential application in vaccines has escalated since the early 1990's. The primary objective is to generate influenza virus from plasmid constructs that can be transfected into a broad range of host cells to provide high viral yields with minimum selection from helper virus. In vivo synthesis of vRNA-like molecules was introduced by using RNA polymerase I (Pol I) dependent transcription of viral RNA (24, 37). In a typical plasmid construct, influenza cDNA is inserted precisely between the murine Pol I promoter and terminator sequences. Upon transfection, vRNA synthesized in the cells is bound by influenza polymerase and nucleoprotein that are provided by helper viruses. However, one major disadvantage in this technique is the cumbersome process of selecting recombinant influenza from the mixture containing the helper viruses. By combining intracellular synthesis of vRNAs and proteins, two reverse genetics systems free of helper virus were established by co-transfection of 12-17 plasmids (9, 23). Both systems utilize eight plasmids to encode vRNAs and four plasmids to encode three viral polymerase subunits and a nucleoprotein. The addition of plasmids expressing the remaining viral structural proteins led to a substantial increase in virus production. Thus, limiting the number of plasmid constructs to generate influenza virus still remained a challenge.


The “ambisense” approach that utilizes two promoters on a bidirectional transcription vector is the first major breakthrough to reduce the number of plasmids required for virus generation (11). In this approach, a Pol I promoter drives the synthesis of vRNA from a cDNA template, whereas, RNA polymerase II (Pol II) promoter drives the synthesis of mRNA from the same template in the opposite direction. A system with eight plasmids (i.e., an eight-plasmid system) was developed using the dual promoter technique, which successfully recovered influenza virus from Vero cells (11). A unidirectional Pol I-Pol II transcription system was also reported, however, it suffers from lower viral yield (11). A much-improved method is the generation of influenza virus using a three-plasmid based reverse genetics system (22). Here, one plasmid carries eight Pol I promoter-driven vRNA transcribing cassettes, another plasmid encodes the three viral polymerase subunits and the third plasmid encodes the nucleoprotein. This three-plasmid system, although arduous to construct, yields higher titers of influenza virus than any of the earlier approaches (22). Use of this technique to generate seed for influenza vaccine would thus require two plasmids individually providing HA and NA from epidemic virus, and three plasmid constructs together to provide the remaining components, making it a “2+3” approach.


Vaccines are necessary to prevent influenza outbreaks. To date, the inactivated and attenuated influenza vaccines commercially available for humans are administered either by injection or by nasal-spray. Influenza vaccine seeds are generated by DNA constructs based on reverse genetics system using the “2+6” strategy, where the HA and NA segments are taken from the circulating strain of influenza virus and the remaining 6 structural segments are taken from either the high productive strain PR8 (A/PR/8/34) or the cold-adapted strain (e.g. A/AA/6/60) (4, 10, 12). The current technology in making influenza vaccines relies on using embryonated eggs, which is time-consuming (takes up to four months), has low viral yield and is a cumbersome procedure.


Use of bacterial species to deliver plasmid DNA encoding viral components in the target host cell is an economical and less cumbersome approach to develop vaccines against influenza virus. However, the challenge would be to minimize the number of plasmid constructs so that it would be much easier to ensure the down stream processes involved in virus generation in a eukaryotic host cell.


The above-mentioned factors present a strong need for a single plasmid system for generating influenza virus to develop an inexpensive, ease of manufacture, quickly modifiable and needle-free influenza vaccine. The present invention addresses the design of a single expression vector for generation of virus, and a bacterial carrier based virus generation system, which could be used to develop vaccines against corresponding viral diseases.


REFERENCE TO COLOR FIGURES

The application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1 depicts the construction of plasmids carrying either dual or mono-promoter elements and their derivatives. (A) Chicken Pol I promoter (CPI) and murine Pol I terminator (MTI) that together comprise the Pol I promoter-terminator system were cloned into the HindIII and NheI sites on the eukaryotic expression vector pcDNA3.1(−) that carries CMV promoter and BGH terminator sequences to construct a bi-directional dual promoter plasmid pYA4379 (SEQ ID NO:57) or its variant pYA4380 (SEQ ID NO:58) that lacks the CMV promoter. (B) A 720 bp long EGFP (enhanced green fluorescent protein) gene fragment flanked on the 5′ and the 3′ ends with non-translating sequences (NTS) from the M segment of WSN virus was cloned into the AarI sites in pYA4379 (SEQ ID NO:57) and the AarI sites in pYA4380 (SEQ ID NO:58) to construct reporter plasmids pYA4387 and pYA4392, respectively. Plasmid pYA4688 was constructed by replacing CPI with Human Pol I promoter (HPI) in pYA4392. Plasmids are not drawn to scale.



FIG. 2 depicts EGFP synthesis as a measure of protein and vRNA synthesis. Chicken embryonic fibroblasts (CEFs) transfected with pYA4387 (A); CEFs transfected with pYA4392 and four helper plasmids pYA4337 (expressing PB2), pYA4338 (expressing PB1), pYA4339 (expressing PA), and pCAWS-NP (expressing NP) (B); HEK (human embryonic kidney) 293 cells transfected with pYA4688 and four helper plasmids pYA4337 (PB2), pYA4338 (PB1), pYA4339 (PA) and pCAWS-NP (C). Images were taken 24 h post transfection at 100× magnification.



FIG. 3 depicts the “eight-plasmid” system of influenza A virus. Plasmids pYA4383, pYA4384, pYA4385, and pYA4386 were constructed by individually cloning the PB2, PB1, PA and NP genes into pYA4379 (SEQ ID NO: 57). Plasmids pYA4388, pYA4389, pYA4390 and pYA4391 were constructed by individually cloning the HA, NA, M and NS genes into plasmid pYA4380 (SEQ ID NO: 58).



FIG. 4 depicts the generation of the p15A ori based T vector. Boxed sequence depicts the T-overhang resulting from excision of the GFP cassette with AhdI. The T-overhang was generated to facilititate convenient cloning of DNA fragments containing an A-overhang at each 3′ end.



FIG. 5 depicts the step-wise construction of the 8-unit-plasmid pYA4519 (SEQ ID NO: 60). (A) PB2, PB1, PA and NP bi-directional cassettes (CPI and MTI in one direction, and cytomegalovirus (CMV) promoter and bovine growth hormone (BGH) polyA sequence in the other direction) were amplified from pYA4379 (SEQ ID NO:57)-derived plasmids (pYA4383, pYA4384, pYA4385, and pYA4386), and each cassette was individually cloned into a p15A-T vector to obtain four 1-unit plasmids p15A-PB2, p15A-PB1, p15A-PA, and p15A-NP. (B) The NS, M, NA and HA vRNA-transcribing cassettes were amplified from pYA4380 (SEQ ID NO:58)-derived plasmids (pYA4391, pYA4390, pYA4389 and pYA4388) by introducing compatible restriction sites and were each cloned into a 1-unit plasmid to obtain four 2-unit plasmids; p15A-PB2-NS, p15A-PB1-M, p15A-PA-NA, and p15A-NP-HA. (C) Each of the two 4-unit plasmids p15A-PB2-NS-PB1-M and p15A-PA-NA-NP-HA was constructed by fusing transcribing cassettes from two of 2-unit plasmids shown in B. (D) The DNA fragment containing PA-NA-NP-HA vRNA transcribing cassettes was excised using KpnI and NgoMIV and ligated into the compatible sites in the 4-unit plasmid p15A-PB2-NS-PB1-M to obtain a 23.6 kb long 8-unit-plasmid pYA4519 (SEQ ID NO:60) to transcribe the whole set of influenza vRNAs via the chicken RNA polymerase I promoter (CPI) and to synthesize influenza virus RNA polymerase (PB1, PB2, PA) and nucleoprotein (NP) by the cytomegalovirus (CMV) promoter. All constructs carry the p15A ori of replication. Plasmids are not drawn to scale. p15A-PB1, polymerase B1 cDNA cassette cloned in p15A-T vector; p15A-PB2, polymerase B2 cDNA cassette cloned in p15A-T vector, p15A-PA, polymerase A cDNA cassette cloned in p15A-T vector; p15A-NP, nucleoprotein cDNA cassette cloned in p15A-T vector; HA=hemagglutinin, NA=neuraminidase, M=matrix protein, NS=non-structural protein.



FIG. 6 depicts the transfection efficiency of the 8-unit-plasmid. CEFs (A and B) and HEK293 cells (E to F) cells transfected with plasmid pYA4731 (pcDNA-mCherry; A and E) or plasmid pYA4732 (pYA4519-mCherry; B and F). CEFs co-transfected with pYA4732 and pYA4392. Expression of mCherry gene (C) and EGFP gene (D) in CEFs was recorded from the same field. HEK293 cells co-transfected with 2 μg of pYA4732 and pYA4688 (G and H). Expression of EGFP gene (G) and mCherry gene (H) was recorded from the same field. Images were taken 24 h post transfection. Magnification, A to F, 100×; G and H, 200×.



FIG. 7 depicts the 8-unit plasmid pYA4562, which is a derivative of pYA4519 with the addition of DNA nuclear targeting sequence (DTS) from simian virus 40 (SV40), and NF-κB binding sequence.



FIG. 8 depicts Salmonella mediated delivery of EGFP reporter plasmid pYA4336. A. Salmonella carriers showed conditional growth on LB-agar plates supplementing with 50 μg/ml DAP and/or 100 μg/ml DL-alanine. B. The pelleted Salmonella carriers were resuspended in LB broth and incubated at 37° C. overnight (standstill). Then, the bacterial cells were collected by centrifugation and stained with propidium iodide (PI) and SYTO9. The dead cells are stained in red fluorescence. The live cells are in green fluorescence. C. Reporter plasmid pYA4336 which only express EGFP in animal cells. D. Plasmid pYA4336 was delivered into CEFs by different Salmonella carriers. As a control, CEFs were also incubated with a mixture of bacterial carrier χ9052 and 15 μg of pYA4336. Cell nuclei were stained with 4′-6-Diamidino-2-phenylindole (DAPI).



FIG. 9 depicts a restriction digestion analysis of plasmid pYA4519 after continuous passages in Salmonella strains χ9052, χ9834, and χ11018. The passage number is noted above each lane. The first lane (M) contains a DNA marker for size reference (10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3 kb, 2.5 kb, 2 kb and 1.5 kb).



FIG. 10 depicts the 8-unit plasmid pYA4732 (pYA4519-mCherry) and CEFs infected by χ9834 carrying pYA4732. As a control, CEFs were also infected by χ9834 carrying pYA4731. Cell nuclei were stained with 4′-6-Diamidino-2-phenylindole (DAPI).



FIG. 11 depicts a 96-well plate for measuring TCID50 of influenza virus rescued from cocultured CEFs/MDCK (Madin-Darby canine kidney) cells by infection with Salmonella Typhimurium carrying pYA4519 or pYA4562.



FIG. 12 depicts the 8-unit plasmids carrying HA and NA genes from influenza A virus (A/chicken/TX/167280-4/02(H5N3). The chloramphenicol resistance marker (cat) and kanamycin resistance marker (kan) in plasmid pYA4929 (A) were replaced with aroA cassette derived from pYA4784. The resulting plasmid is designated as pYA4930 (B).



FIG. 13 depicts plasmids pYA3681, pYA4594, pYA4589 and pYA4595. These plasmids express both asd and murA genes under the regulation of the araC PBAD activator-promoter.





DETAILED DESCRIPTION OF THE INVENTION

A single expression vector capable of generating an attenuated virus from a segmented genome has been developed. An auxotrophic bacterial carrier can carry and deliver this expression vector into in vitro cultured cells, resulting in the recovery of virus, either attenuated or non-attenuated. The invention greatly simplifies the process of producing viruses that have segmented genomes, which historically have required transfection of multiple expression vectors for vRNA expression, in addition to vectors for expressing mRNAs for translation to viral replication proteins. Advantageously, as illustrated in the examples, the expression vector is stable in bacteria at 37° C., and produces higher titers of virus than traditional multi-vector systems when transfected into eukaryotic cells. This invention also demonstrates that bacterial carrier mediated delivery of such an expression vector can lead to the generation of virus. Therefore, this invention provides a system for bacterial carrier based delivery of attenuated viral vaccines with advantages of low cost, ease of manufacture, flexibility in introducing desired alterations, and finally, needle-free administration.


I. Expression Vector

The expression vector generally comprises a plasmid having at least two types of transcription cassettes. One transcription cassette is designed for vRNA production. The other transcription cassette is designed for the production of both vRNAs, and mRNAs. As will be appreciated by a skilled artisan, the number of transcription cassettes, and their placement within the vector relative to each other, can and will vary depending on the segmented virus that is produced. Each of these components of the expression vector is described in more detail below.


The expression vector may be utilized to produce several different segmented and nonsegmented viruses. Viruses that may be produced from the expression vector include positive-sense RNA viruses, negative-sense RNA viruses and double-stranded RNA (ds-RNA) viruses.


In one embodiment, the virus may be a positive-sense RNA virus. Non-limiting examples of positive-sense RNA virus may include viruses of the family Arteriviridae, Caliciviridae, Coronaviridae, Flaviviridae, Picornaviridae, Roniviridae, and Togaviridae. Non-limiting examples of positive-sense RNA viruses may include SARS-coronavirus, Dengue fever virus, hepatitis A virus, hepatitis C virus, Norwalk virus, rubella virus, West Nile virus, Sindbis virus, Semliki forest virus and yellow fever virus.


In one embodiment, the virus may be a double-stranded RNA virus. Non-limiting examples of segmented double-stranded RNA viruses may include viruses of the family Reoviridae and may include aquareovirus, blue tongue virus, coltivirus, cypovirus, fijivirus, idnoreovirus, mycoreovirus, orbivirus, orthoreovirus, oryzavirus, phytoreovirus, rotavirus, infectious bursal disease virus and seadornavirus.


In yet another embodiment, the virus may be a negative-sense RNA virus. Negative-sense RNA viruses may be viruses belonging to the families Orthomyxoviridae, Bunyaviridae, and Arenaviridae with six-to-eight, three, or two negative-sense vRNA segments, respectively. Non-limiting examples of negative-sense RNA viruses may include thogotovirus, isavirus, bunyavirus, hantavirus, nairovirus, phlebovirus, tospovirus, tenuivirus, ophiovirus, arenavirus, deltavirus and influenza virus.


In another aspect, the invention provides an expression vector capable of generating influenza virus. There are three known genera of influenza virus: influenza A virus, influenza B virus and influenza C virus. Each of these types of influenza viruses may be produced utilizing the single expression vector of the invention.


In one exemplary embodiment, the expression vector is utilized to produce Influenza A virus. Influenza A viruses possess a genome of 8 vRNA segments, including PA, PB1, PB2, HA, NP, NA, M and NS, which encode a total of ten to eleven proteins. To initiate the replication cycle, vRNAs and viral replication proteins must form viral ribonucleoproteins (RNPs). The influenza RNPs consist of the negative-sense viral RNAs (vRNAs) encapsidated by the viral nucleoprotein, and the viral polymerase complex, which is formed by the PA, PB1 and PB2 proteins. The RNA polymerase complex catalyzes three different reactions: synthesis of an mRNA with a 5′ cap and 3′ polyA structure essential for translation by the host translation machinery; a full length complementary RNA (cRNA), and of genomic vRNAs using the cRNAs as a template. Newly synthesized vRNAs, NP and, PB1, PB2 and PA polymerase proteins are then assembled into new RNPs, for further replication or encapsidation and release of progeny virus particles. Therefore, to produce influenza virus using a reverse genetics system, all 8 vRNAs and mRNAs that express the viral proteins essential for replication (NP, PB1, PB1 and PA), must be synthesized. The expression vector of the invention may be utilized to produce all of these vRNAs and mRNAs.


The expression vector may also be utilized to produce any serotype of influenza A virus without departing from the scope of the invention. Influenza A viruses are classified into serotypes based upon the antibody response to the viral surface proteins hemagglutinin (HA or H) encoded by the HA vRNA segment, and neuraminidase (NA or N) encoded by the NA vRNA segment. At least sixteen H subtypes (or serotypes) and nine N subtypes of influenza A virus have been identified. New influenza viruses are constantly being produced by mutation or by reassortment of the 8 vRNA segments when more than one influenza virus infects a single host. By way of example, known influenza serotypes may include H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2, and H10N7 serotypes.


(a) Vector

The expression vector of the invention comprises a vector. As used herein, “vector” refers to an autonomously replicating nucleic acid unit. The present invention can be practiced with any known type of vector, including viral, cosmid, phasmid, and plasmid vectors. The most preferred type of vector is a plasmid vector. As is well known in the art, plasmids and other vectors may possess a wide array of promoters, multiple cloning sequences, and transcription terminators.


The vector may have a high copy number, an intermediate copy number, or a low copy number. The copy number may be utilized to control the expression level for the transcription cassettes, and as a means to control the expression vector's stability. In one embodiment, a high copy number vector may be utilized. A high copy number vector may have at least 31, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 copies per bacterial cell. In other embodiments, the high copy number vector may have at least 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 copies per bacterial cell. Non-limiting examples of high copy number vectors may include a vector comprising the pBR ori or the pUC ori. In an alternative embodiment, a low copy number vector may be utilized. For example, a low copy number vector may have one or at least two, three, four, five, six, seven, eight, nine, or ten copies per bacterial cell. A non-limiting example of low copy number vector may be a vector comprising the pSC101 ori. In an exemplary embodiment, an intermediate copy number vector may be used. For instance, an intermediate copy number vector may have at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 copies per bacterial cell. A non-limiting example of an intermediate copy number vector may be a vector comprising the p15A ori.


The vector may further comprise a selectable marker. Generally speaking, a selectable marker encodes a product that the host cell cannot make, such that the cell acquires resistance to a specific compound or is able to survive under specific conditions. For example, the marker may code for an antibiotic resistance factor. Suitable examples of antibiotic resistance markers include, but are not limited to, those coding for proteins that impart resistance to kanamycin, spectomycin, neomycin, gentamycin (G418), ampicillin, tetracycline, and chlorampenicol. However, use of selective markers for drug resistance is undesirable for live attenuated bacterial vaccines and delivery systems and is also undesirable for DNA vaccines. Thus in still other cases, the vector might preferably have selectable Asd+, MurA+, AroA+, DadB+, Alr+, AroC+, AroD+, IlvC+ and/or IlvE+ when the expression vector is used in a balanced-lethal or balanced-attenuation vector-host system when present in and delivered by carrier bacteria.


In some embodiments, the vector may also comprise a transcription cassette for expressing non-viral reporter proteins. By way of example, reporter proteins may include a fluorescent protein, luciferase, alkaline phosphatase, beta-galactosidase, beta-lactamase, horseradish peroxidase, and variants thereof.


In some embodiments, the vector may also comprise a DNA nuclear targeting sequence (DTS). A non-limiting example of a DTS may include the SV40 DNA nuclear targeting sequence.


In some embodiments, the vector may also comprise a NF-κB binding site. The SV40 DTS and NF-κB binding sequence facilitate nuclear import of the plasmid DNA, and this facilitates transcription of genetic sequences on the vector.


(b) Transcription Cassettes for vRNAs Expression


The expression vector comprises at least one transcription cassette for vRNA production. Generally speaking, the transcription cassette for vRNA production minimally comprises a Pol I promoter operably linked to a viral cDNA linked to a Pol I transcription termination sequence. In an exemplary embodiment, the transcription cassette will also include a nuclear targeting sequence. The number of transcription cassettes for vRNA production within the expression vector can and will vary depending on the virus that is produced. For example, the expression vector may comprise two, three, four, five, six, seven, or eight or more transcription cassettes for vRNA production. When the virus that is produced is influenza, the expression cassette typically will comprise four transcription cassettes for vRNA production.


The term “viral cDNA”, as used herein, refers to a copy of deoxyribonucleic acid (cDNA) sequence corresponding to a vRNA segment of an RNA virus genome. cDNA copies of viral RNA segments may be derived from vRNAs using standard molecular biology techniques known in the art (see, e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual,” 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, and Knipe et al (2006) “Fields Virology”, Fifth Edition, Lippincott Williams & Wilkins (2007). In some embodiments, the cDNA may be derived from a naturally occurring virus strain or a virus strain commonly used in vitro. In other embodiments, the cDNA may be derived synthetically by generating the cDNA sequence in vitro using methods known in the art. The natural or synthetic cDNA sequence may further be altered to introduce mutations and sequence changes. By way of example, a naturally occurring viral sequence may be altered to attenuate a virus, to adapt a virus for in vitro culture, or to tag the encoded viral proteins.


The selection of promoter can and will vary. The term “promoter”, as used herein, may mean a synthetic or naturally derived molecule that is capable of conferring, activating or enhancing expression of a nucleic acid. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of a nucleic acid. The term “operably linked,” as used herein, may mean that expression of a nucleic acid is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5′ (upstream) of the nucleic acid under its control. The distance between the promoter and a nucleic acid to be expressed may be approximately the same as the distance between that promoter and the nucleic acid sequence it controls. As is known in the art, variation in this distance may be accommodated without loss of promoter function. The promoters may be of viral, prokaryotic, phage or eukaryotic origin. Non-limiting examples of promoters may include T7 promoter, T3 promoter, SP6 promoter, RNA polymerase I promoter and combinations thereof. In some embodiments, the promoters may be different in each transcription cassette. In preferred embodiments, the promoters may be the same in each transcription cassette. In preferred alternatives of this embodiment, the promoters may be RNA polymerase I (Pol I) promoters. In an exemplary alternative of this embodiment, the promoters may be human Pol I promoters. In another exemplary alternative of this embodiment, the promoters may be chicken Pol I promoters. In a further exemplary alternative of this embodiment, the promoters are human Pol I promoters as described in Example 1. In another exemplary alternative of this embodiment, the promoters are chicken Pol I promoters as described in Example 1.


The promoter may be operably linked to the cDNA to produce a negative-sense vRNA or a positive-sense cRNA. In an exemplary alternative of this embodiment, the promoter may be operably linked to the cDNA to produce a negative-sense vRNA.


The transcription cassette also includes a terminator sequence, which causes transcriptional termination at the end of the viral cDNA sequence. By way of a non-limiting example, terminator sequences suitable for the invention may include a Pol I terminator, the late SV40 polyadenylation signal, the CMV polyadenylation signal, the bovine growth hormone polyadenylation signal, or a synthetic polyadenylation signal. In some embodiments, the terminators may be different in each transcription cassette. In a preferred embodiment, the terminators may be the same in each transcription cassette. In one alternative of this embodiment, the Pol I terminator may be a human Pol I terminator. In an exemplary embodiment, the terminator is a murine Pol I terminator. In an exemplary alternative of this embodiment, the terminator sequence of the expression cassettes may be a truncated version of the murine Pol I terminator as described in Example 1.


To function properly during replication, vRNAs transcribed from the transcription cassettes generally have precise 5′ and 3′ ends that do not comprise an excess of non-virus sequences. Depending on the promoters and terminators used, this may be accomplished by precise fusion to promoters and terminators or, by way of example, the transcription cassette may comprise ribozymes at the ends of transcripts, wherein the ribozymes cleave the transcript in such a way that the sequences of the 5′ and 3′ termini are generated as found in the vRNA.


As will be appreciated by a skilled artisan, when the expression vector produces influenza virus, the expression vector may comprise at least one transcription cassette for vRNA production. The transcription cassette may be selected from the group consisting of (1) a Pol I promoter operably linked to an influenza virus HA cDNA linked to a Pol I transcription termination sequence; (2) a Pol I promoter operably linked to an influenza virus NA cDNA linked to a Pol I transcription termination sequence; (3) a Pol I promoter operably linked to an influenza virus M cDNA linked to a Pol I transcription termination sequence; and (4) a Pol I promoter operably linked to an influenza virus NS cDNA linked to a Pol I transcription termination sequence. The expression vector may comprise at least 2, 3, or 4 of these transcription cassettes. In an exemplary embodiment, the expression vector will also include either one or two different nuclear targeting sequences (e.g., SV40 DTS and NF-κB binding sequence).


In an exemplary embodiment when the expression vector produces influenza virus, the expression vector will comprise four transcription cassettes for vRNA production. The transcription cassettes for this embodiment will comprise (1) a Pol I promoter operably linked to an influenza virus HA cDNA linked to a Pol I transcription termination sequence; (2) a Pol I promoter operably linked to an influenza virus NA cDNA linked to a Pol I transcription termination sequence; (3) a Pol I promoter operably linked to an influenza virus M cDNA linked to a Pol I transcription termination sequence; and (4) a Pol I promoter operably linked to an influenza virus NS cDNA linked to a Pol I transcription termination sequence. In an exemplary embodiment, the expression vector will also include either one or two different nuclear targeting sequences (e.g., SV40 DTS and NF-κB binding sequence).


(c) Transcription Cassettes for vRNA and mRNA Expression


The expression vector comprises at least one transcription cassette for vRNA and mRNA production. Typically, the transcription cassette for vRNA and mRNA production minimally comprises a Pol I promoter operably linked to a viral cDNA linked to a Pol I transcription termination sequence, and a Pol II promoter operably linked to the viral cDNA and a Pol II transcription termination sequence. In an exemplary embodiment, the transcription cassette will also include a nuclear targeting sequence. The number of transcription cassettes for vRNA and mRNA production within the expression vector can and will vary depending on the virus that is produced. For example, the expression vector may comprise two, three, four, five, six, seven, or eight or more transcription cassettes for vRNA and mRNA production. When the virus that is produced is influenza, the expression cassette typically may comprise four transcription cassettes for vRNA and mRNA production.


The viral cDNA, Pol I promoter and Pol I terminator suitable for producing vRNA is as described above in section (b).


For mRNA production, each transcription cassette comprises a Pol II promoter operably linked to cDNA and a Pol II termination sequence. Non-limiting examples of promoters may include the cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, simian virus 40 (SV40) early promoter, ubiquitin C promoter or the elongation factor 1 alpha (EF1α) promoter. In some embodiments, the promoters may be different in each transcription cassette. In preferred embodiments, the promoters may be the same in each transcription cassette. In preferred alternatives of this embodiment, the promoters may be the CMV Pol II promoter. In an exemplary alternative of this embodiment, the promoters are CMV Pol II promoters as described in Example 1.


Each transcription cassette also comprises a Pol II terminator sequence. By way of non-limiting example, terminator sequences suitable for the invention may include the late SV40 polyadenylation signal, the CMV polyadenylation signal, the bovine growth hormone (BGH) polyadenylation signal, or a synthetic polyadenylation signal. In some embodiments, the terminators may be different in each transcription cassette. In a preferred embodiment, the terminators may be the same in each transcription cassette. In an exemplary embodiment, the terminator is a BGH polyadenylation signal. In an exemplary alternative of this embodiment, the terminator sequence of the expression cassettes may be a truncated version of the BGH polyadenylation signal as described in Example 1.


To function properly in initiating vRNA replication, mRNAs transcribed from the transcription cassettes may contain signals for proper translation by the host cell translation machinery. Most cellular mRNAs transcribed from a Pol II promoter are capped at the 5′ end and polyadenylated at the 3′ end after transcription to facilitate mRNA translation. However, some cellular mRNAs and many viral mRNAs encode other sequences that facilitate translation of the mRNA in the absence of a 5′ cap structure or 3′ polyA structure. By way of example, some cellular mRNAs and viral mRNAs may encode an internal ribosomal entry site (IRES), which could functionally replace the 5′ cap. By way of another example, some mRNAs and viral mRNAs may encode an RNA structure, such as a pseudoknot, at the 3′ end of the mRNA, which could functionally replace the 3′ polyA. In an exemplary embodiment, the mRNAs transcribed from the transcription cassettes are capped at the 5′ end and polyadenylated at the 3′ end.


As will be appreciated by a skilled artisan, when the expression vector produces influenza virus, the expression vector may comprise at least one transcription cassette for vRNA and mRNA production. The transcription cassette may be selected from the group consisting of (1) a Pol I promoter operably linked to an influenza virus PA cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PA cDNA and a Pol II transcription termination sequence; (2) a Pol I promoter operably linked to an influenza virus PB1 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB1 cDNA and a Pol II transcription termination sequence; (3) a Pol I promoter operably linked to an influenza virus PB2 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB2 cDNA and a Pol II transcription termination sequence; and (4) a Pol I promoter operably linked to an influenza virus NP cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the NP cDNA and a Pol II transcription termination sequence. The expression vector may comprise at least 2, 3, or 4 of these transcription cassettes. In an exemplary embodiment, the expression vector will also include either one or two different nuclear targeting sequences (e.g., SV40 DTS or NF-κB binding sequence).


In an exemplary embodiment when the expression vector produces influenza virus, the expression vector will comprise four transcription cassettes for vRNA and mRNA production. The transcription cassettes for this embodiment will comprise (1) a Pol I promoter operably linked to an influenza virus PA cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PA cDNA and a Pol II transcription termination sequence; (2) a Pol I promoter operably linked to an influenza virus PB1 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB1 cDNA and a Pol II transcription termination sequence; (3) a Pol I promoter operably linked to an influenza virus PB2 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB2 cDNA and a Pol II transcription termination sequence; and (4) a Pol I promoter operably linked to an influenza virus NP cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the NP cDNA and a Pol II transcription termination sequence. In an exemplary embodiment, each expression plasmid construct will also include either one or two different nuclear translocation signals (e.g., SV40 DTS or NF-κB binding sequence).


(d) Exemplary Expression Vectors

In an exemplary iteration of the invention, a single expression vector will comprise all of the genomic segments necessary for the production of influenza virus in a host cell. As detailed above, for the production of influenza virus HA, NA, NS, and M vRNA must be produced and PA, PB1, PB2, and NP vRNA and mRNA must be produced. For this iteration, the expression vector will comprise four transcription cassettes for vRNA production and four transcription cassettes for vRNA and mRNA production. The four cassettes for vRNA production will comprise (1) a Pol I promoter operably linked to an influenza virus HA cDNA linked to a Pol I transcription termination sequence; (2) a Pol I promoter operably linked to an influenza virus NA cDNA linked to a Pol I transcription termination sequence; (3) a Pol I promoter operably linked to an influenza virus M cDNA linked to a Pol I transcription termination sequence; and (4) a Pol I promoter operably linked to an influenza virus NS cDNA linked to a Pol I transcription termination sequence. The four transcription cassettes for vRNA and mRNA production will comprise (1) a Pol I promoter operably linked to an influenza virus PA cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PA cDNA and a Pol II transcription termination sequence; (2) a Pol I promoter operably linked to an influenza virus PB1 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB1 cDNA and a Pol II transcription termination sequence; (3) a Pol I promoter operably linked to an influenza virus PB2 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB2 cDNA and a Pol II transcription termination sequence; and (4) a Pol I promoter operably linked to an influenza virus NP cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the NP cDNA and a Pol II transcription termination sequence. The expression vector will preferably also include either one or two different nuclear translocation signals (e.g., SV40 DTS or NF-κB binding sequence). In an exemplary embodiment, the vector is a plasmid. The plasmid will generally be a low or intermediate copy number plasmid. A particularly exemplary expression vector for this embodiment is detailed in the Examples.


The arrangement and direction of transcription cassettes within the single expression vector relative to each other can and will vary without departing from the scope of the invention. It is believed, however, without being bound by any particular theory that arrangement of transcription cassettes in pairs of vRNA cassettes and vRNA and mRNA cassettes is preferable because it may reduce the degree of recombination and as a result, yield an expression vector with increased genetic stability.


It is also envisioned that in certain embodiments, influenza genomic segments may be produced from more than a single expression vector without departing from the scope of the invention. The genomic segments may be produced, for example, from 2, 3, or 4 or more different expression vectors. In an iteration of this embodiment, NS, and M vRNA, and PA, PB1, PB2, and NP vRNA and mRNA are produced from a single expression vector. For this iteration, the expression vector will comprise two transcription cassettes for vRNA production and four transcription cassettes for vRNA and mRNA production. The two transcription cassettes for vRNA production will comprise (1) a Pol I promoter operably linked to an influenza virus M cDNA linked to a Pol I transcription termination sequence; and (2) a Pol I promoter operably linked to an influenza virus NS cDNA linked to a Pol I transcription termination sequence. The four transcription cassettes for vRNA and mRNA production will comprise (1) a Pol I promoter operably linked to an influenza virus PA cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PA cDNA and a Pol II transcription termination sequence; (2) a Pol I promoter operably linked to an influenza virus PB1 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB1 cDNA and a Pol II transcription termination sequence; (3) a Pol I promoter operably linked to an influenza virus PB2 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB2 cDNA and a Pol II transcription termination sequence; and (4) a Pol I promoter operably linked to an influenza virus NP cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the NP cDNA and a Pol II transcription termination sequence. The expression of HA vRNA and NA vRNA may be from a single expression vector that comprises two transcription cassettes comprising (1) a Pol I promoter operably linked to an influenza virus HA cDNA linked to a Pol I transcription termination sequence; and (2) a Pol I promoter operably linked to an influenza virus NA cDNA linked to a Pol I transcription termination sequence. Alternatively, expression of HA vRNA and NA vRNA may be from two separate expression vectors.


In some embodiments, restriction digestion sites may be placed at convenient locations in the expression vector. By way of example, restriction enzyme sites placed at the extremities of the cDNAs may be used to facilitate replacement of cDNA segments to produce a desired reassortment or strain of the virus. By way of another example, restriction enzyme sites placed at the extremities of the transcription cassettes may be used to facilitate replacement of transcription cassettes to produce a desired reassortment or strain of the virus. Suitable, endonuclease restriction sites include sites that are recognized by restriction enzymes that cleave double-stranded nucleic acid. By way of non-limiting example, these sites may include AarI, AccI, AgeI, Apa, BamHI, BglI, BglII, BsiWI, BssHI, BstBI, ClaI, CviQI, Ddel, DpnI, DraI, EagI, EcoRI, EcoRV, FseI, FspI, HaeII, HaeIII, HhaI, HincII, HindIII, HpaI, HpaII, KpnI, KspI, MboI, MfeI, NaeI, NarI, NcoI, NdeI, NgoMIV, NheI, NotI, PacI, PhoI, PmlI, PstI, PvuI, PvulI, SacI, SacII, SalI, SbfI, SmaI, SpeI, SphI, SrfI, StuI, TaqI, TfiI, TliI, XbaI, XhoI, XmaI, XmnI, and ZraI. In an exemplary alternative of this embodiment, the restriction enzyme site may be AarI.


II. Bacterial Carrier

An additional aspect of the invention comprises a bacterial carrier that can carry and deliver the expression vector described in Section I into a host cell. The host cell may be in vitro (i.e., cultured cells) or in vivo (e.g., an animal) as described in more detail in section III below. The bacterial carrier is typically auxotrophic and may be either a Gram-positive bacterium or Gram-negative bacterium. In this context, the bacterial carrier generally carries at least one gene mutation for an auxotrophic phenotype to enable intracellular release of the expression vector, and at least one gene mutation to enable stable carriage of the expression vector and at least one mutation to impose appropriate attenuation and for other desirable phenotypes such as for escaping the endosome in a eukaryotic cell. Additionally, the bacterial carrier may be a live bacterium or a bacterial ghost. In addition, the bacterial carrier may be attenuated. The bacterial carrier may also carry additional plasmid vectors for better invasion efficiency or for regulated delayed lysis in vivo. Preferably, the bacterial carrier is sensitve to all antimicrobial drugs including antibiotics that might be useful in treating infections with wild-type variants of the particular bacterial carrier being used to deliver the plasmid vector to eukaryotic cells.


As will be appreciated by a skilled artisan, the bacterial carrier may be utilized to deliver a single expression vector or to deliver multiple expression vectors. The single expression vector may encode information for generation of a segmented virus or non-segmented virus; for instance, the expression vector can encode 8 vRNAs, 3 polymerase subunits and nucleoprotein of influenza virus.


Alternatively, the bacterial carrier may be utilized to deliver multiple expression vectors. For example, one p15A ori based expression vector encodes PB2, PB1, PA and NP genes, and the other pBR ori based expression vector encodes HA, NA, M and NS genes.


In yet another embodiment, the bacterial carrier may be utilized to deliver an expression vector for virus generation. For example, the expression vector pYA4519 encodes 8 vRNAs, 3 polymerase subunits and nucleoprotein of influenza virus.


In one embodiment, the bacterial carrier may be utilized to deliver an expression vector in vitro. For instance, the expression vector encodes 8 vRNAs, 3 polymerase subunits and nucleoprotein of influenza virus.


In an alternative embodiment, the bacterial carrier may be utilized to deliver an expression vector in vivo. For example, oral administration with an auxotrophic, attenuated Salmonella Typhimurium carrying pYA4930 designed for regulated delayed lysis to deliver pYA4930 into avians.


In one embodiment, the bacterial carrier may be utilized to deliver an expression vector to humans. By way of non-limiting example, the expression vector encodes HA and NA from epidemic influenza virus, and the other 6 segments from cold-adapted influenza virus (e.g. A/AA/6/60). The polybasic cleavage site in HA will be removed to avoid the generation of reassortant virulent virus in the host. In this embodiment, the vRNAs transcription is regulated by human RNA Pol I promoters, and the transcription of mRNAs is regulated by CMV promoters.


In another embodiment, the bacterial carrier may be utilized to deliver expression vectors into other animals. For example, the expression vector encodes HA and NA from a highly pathogenic avian influenza virus (polybasic cleavage site in HA will be removed to avoid the generation of reassortant virulent virus in the host), and the other 6 segments from a cold-adapted influenza virus (e.g. A/AA/6/60).


In each of the foregoing embodiments, the bacterial carrier may be designed to have host-specificity for and be utilized for primates (e.g., humans, monkeys, chimpanzies etc), poultry (e.g., chickens, turkeys, ducks, geese and other fowl), ruminants (e.g., beef cattle, dairy cattle, and sheep, etc), pigs, and companion animals (e.g., horses, dogs, cats, and other pets).


As will be appreciated by a skilled artisan, suitable bacterial carriers may comprise several different bacterial strains to the extent the bacterial strain is capable of maintaining and delivering an expression vector to a host cell. By way of non-limiting example, the bacterial strain may be Gram-negative bacteria, including Salmonella spp., Shigella spp, Yersinia spp., and engineered Escherichia coli expressing an invasin gene. In a preferred alternative of this embodiment, the bacterium may be a Salmonella enterica serovar. In one alternative of this embodiment, the bacterium may be a Salmonella enterica serovar Abortusovis. In another alternative of this embodiment, the Salmonella bacterium may be Salmonella enterica serovar Typhi. In a preferred embodiment, the bacterium may be a Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). In an exemplary alternative of this embodiment, the Salmonella Typhimurium strain is χ9052 (ΔasdA33 Δalr-3 ΔdadB4). In other exemplary alternatives of this embodiment, the Salmonella Typhimurium strain is χ11017 (ΔasdA27::TT araC PBAD c2 ΔaraBAD23 Δ(gmd-fcl)-26 Δpmi-2426 ΔrelA198::TT araC PBAD lacI ΔPmurA25::araC PBAD murA) or χ11327 (ΔasdA27::TT araC PBAD c2 ΔPmurA25::TT araC PBAD murA ΔaraBAD23 Δ(gmd-fcl)-26 ΔrelA198::araC PBAD lacI TTΔpmi-2426 ΔtlpA181 ΔsseL116 ΔPhilA::Ptrc ΔlacO888 hilA ΔsifA26).


In an alternative of this embodiment, the Salmonella Typhimurium strains may also comprise deletions of the bacterial nucleic acid sequences recA62, recF126 or both. In an alternative of this embodiment, the Salmonella Typhimurium strains may also comprise a deletion of the bacterial nucleic acid sequence for the aroA gene to result in the aroA21419 mutation.


Alternatively, the bacterial strain may be Gram-positive bacteria. By way of non-limiting example, one suitable Gram-positive bacterium is Listeria monocytogenes.


In certain embodiments, the bacterial carrier may be attenuated. By way of example, the bacterial carrier may be live bacteria with appropriate attenuation due to a phoP mutation or other means of attenuation if the carrier is derived from a pathogenic bacterium capable of causing disease. In yet another embodiment, the bacterial carrier may be bacteria with a regulated delayed lysis genotype, such as araC PBAD promoter regulated expression of the murA gene. The live bacteria carrying an expression vector may be induced to express a phage lysis gene E or some other lysis gene to form bacterial ghosts.


In an alternative embodiment, the bacterial carrier may carry a mutation in at least one gene for an auxotrophic phenotype. For example, these genes include, but are not limited to aroA, aroC, aroD, llvC, llvE, asd, murA, dadB, and alr.


In certain embodiments to facilitate stable carriage of an expression vector with repetitive sequences, either recA or recF gene inactivation may be included to reduce either intra- or inter-plasmid recombination.


In certain embodiments the bacterial carrier may carry a sifA mutation to facilitate escape from the endosome.


In other embodiments the bacterial carrier may carry an endA mutation to minimize chances of endonuclease digestion of the expression vector.


Several methods generally known in the art utilized to attenuate a bacterial carrier may be employed without departing from the scope of the invention. Suitable non-limiting examples of such attenuation means include gene mutations in phoP, phoQ, cya, crp, cdt, an aro gene, asd, a dap gene, dadB and alr, murA, nadA, pncB, rpsL, ilvE, rpoS, ompR, htrA, rfc, poxA, dam, hemA, sodC, recA, ssrA, sirA, inv, hilA, rpoE, flgM, tonB, slyA, pmi, galE, galU, mviA, rfaH, a pur gene, a pab gene, and fur.


In a further embodiment, the bacterial carrier may also comprise additional plasmid vectors for improving its invasion efficiency. For example, a plasmid expressing the gene encoding invasin from Yersinia pseudotuberculosis.


In an additional embodiment, the bacterial carrier may comprise additional plasmid vectors for regulated lysis in vivo. For example, the plasmid pYA3681 (araC PBAD promoter regulates expression of asd and murA genes) in strain χ11020.


III. Methods for Producing a Segmented Virus

The expression vector detailed in section (I) may be utilized to produce a segmented virus in vitro or in vivo. Depending upon the intended use, the resulting virus may, by way of example, be purified, attenuated or inactivated. In some embodiments, the virus is purified and used as a seed virus for further production of virus. In other embodiments, the virus is attenuated for use in a vaccine composition. In yet other embodiments, the virus is inactivated for use in a vaccine composition.


In one aspect, the invention provides a method for producing a virus by introducing the expression vector into a eukaryotic cell. The expression vector may be delivered to the cell using transfection. Methods for transfecting nucleic acids are well known to individuals skilled in the art. Transfection methods include, but are not limited to, cationic transfection, liposome transfection, dendrimer transfection, electroporation, heat shock, nucleofection transfection, magnetofection, nanoparticles, biolistic particle delivery (gene gun), and proprietary transfection reagents such as Lipofectamine, Dojindo Hilymax, Fugene, jetPEI, Effectene, DreamFect, or ExGen 500.


The expression vector may also be delivered to the cell using a viral vector. Viral vectors suitable for introducing nucleic acids into cells include retroviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, rhabdoviruses, and herpes viruses.


In some embodiments, the expression vector may be introduced into eukaryotic tissue culture cells in vitro. Non-limiting examples of eukaryotic cells used for virus production in vitro may include human embryonic kidney 293 (HEK293) cells, Madin-Darby canine kidney (MDCK) cells, chicken embryonic fibroblasts (CEFs), African green monkey kidney epithelial (vero) cells, or any variants or combinations thereof. In all such cases, the sequences in all expression cassettes recognized by RNA polymerase I would have to be changed to possess DNA sequences recognized by the RNA polymerase I from the species of animal for the particular cell line. This is because RNA polymerase I are species specific. In a preferred embodiment, the expression vector may be introduced into HEK293 cells. In another preferred embodiment, the expression vector may be introduced into a mixture of CEFs and MDCK cells. Upon introduction of the expression vector into the eukaryotic cells, the host cells may then be cultured under conditions that permit production of viral proteins and vRNAs using tissue culture techniques known in the art. By way of non-limiting example, the expression vector, when introduced into a tissue culture cell, yields 108 PFU/ml or more of influenza virus after 6 days.


In other aspects, the expression vector may be introduced into a eukaryotic cell in an animal. Non-limiting examples of animals where the expression vector may be introduced may include humans, horses, pigs, chickens, ducks, and geese. Methods of delivery of the expression vector to a eukaryotic cell may be as described above.


Alternatively, and in a preferred embodiment of the invention, the expression vector may be delivered into the eukaryotic cell via a carrier bacterium as described in Section II. The carrier bacteria typically deliver the expression vector into the eukaryotic cell cytoplasm. Suitable carrier bacteria are described in more detail in Section II.


In yet other aspects, bacterial carrier mediated expression vector delivery can be used to generate several different groups of viruses, including positive-sense RNA viruses, negative-sense RNA viruses and double-stranded RNA (ds-RNA) viruses. Non-limiting examples of positive-sense RNA virus include viruses of the family Arteriviridae, Caliciviridae, Coronaviridae, Flaviviridae, Picornaviridae, Roniviridae, and Togaviridae. Non-limiting examples of positive-sense RNA viruses may include SARS-coronavirus, Dengue fever virus, hepatitis A virus, hepatitis C virus, Norwalk virus, rubella virus, West Nile virus, Sindbis virus, Semliki forest virus and yellow fever virus. Non-limiting examples of double-stranded RNA viruses may include viruses of the family Reoviridae and may include aquareovirus, coltivirus, cypovirus, fijivirus, idnoreovirus, mycoreovirus, orbivirus, orthoreovirus, oryzavirus, phytoreovirus, rotavirus, infectious bursal disease virus and seadornavirus. Negative-sense RNA viruses may be viruses belonging to the families Orthomyxoviridae, Bunyaviridae, and Arenaviridae with six-to-eight, three, or two negative-sense vRNA segments respectively. Non-limiting examples of negative-sense RNA viruses may include thogotovirus, isavirus, bunyavirus, hantavirus, nairovirus, phlebovirus, tospovirus, tenuivirus, ophiovirus, arenavirus, deltavirus and influenza virus.


In some embodiments, the bacterial carriers are attenuated as detailed in Section II. As previously described, the bacterial carrier may carry one or more mutations for this purpose. Non-limiting examples are the phoP mutation and the pmi mutation. The bacterial carrier may carry one plasmid to express a lysis gene. Non-limiting example is phage lysis gene E expressing plasmid. The bacterial carrier may carry one plasmid, which complement the mutations on the bacterial carrier chromosome to form a regulated delayed lysis system. For example, χ11020 carrying plasmid pYA3681.


In some embodiments, the expression vector may be modified for generation of attenuated virus. The strategies include, but not limiting to (1) using an attenuated virus genome to construct the single expression vector. For example, using HA and NA from epidemic influenza virus and the other segments from attenuated cold-adapted influenza virus (e.g. A/AA/6/60). Meanwhile the polybasic cleavage site has to be removed from the HA protein. (2) Introducing mutations into viral genes to change the protein sequence. For example, introducing mutations into epidemic influenza virus by reverse genetics to attenuate it, so that the generated virus can be used as vaccine seed. The mutations include (i) removing the polybasic cleavage site from HA protein, (ii) truncating the C-terminal end of the NS1 protein, (iii) and introducing mutations into viral polymerase.


DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.


The term “cRNA” refers to a positive-sense RNA copy of a vRNA.


The term “vRNA” refers to a negative-sense genomic viral RNA.


The term “vaccine composition” as used herein means a composition that when administered to a host, typically elicits an immune response against the virus. Such compositions are known in the art.


Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.


Materials and Methods for Examples 1-4

Bacterial strains, enzymes, plasmids and primers. EPI300™ chemically competent E. coli (Epicentre) was used for all DNA cloning experiments. Restriction enzyme SrfI was bought from Stratagene (La Jolla, Calif.). All other restriction enzymes were from New England Biolabs (Ipswich, Mass.). Plasmids pTM-Pol I-WSN-All and pCAWS-NP were kindly provided by Dr. Yoshihiro Kawaoka (University of Wisconsin—Madison). Plasmid pYS1190 and pIRES-EGFP were gifts from Dr. Yixin Shi (Arizona State University). Primers used in this study are listed in Table 2. Plasmid constructs used in this study are listed in Table 1.









TABLE 1







Plasmid constructs used in this study.









Plasmid
Properties
Reference





pcDNA3.1(-)
Eukaryotic expression vector carrying a CMV promoter and bovine
Invitrogen



growth hormone polyadenylation signal


pIRES-EGFP
Source of the EGFP gene
Clontech


pYS1190
Source of the mCherry gene
Unpublished


pTM-PolI-WSN-All
An 8-unit-plasmid for transcribing PB1, PB2, NS, M, NA, PA, NP, HA vRNAs
-22



under human Pol I promoter


pCAWS-NP
Eukaryotic expression of nucleoprotein (NP) used as helper plasmid
-22


pYA3994
A pBR ori containing plasmid containing GFP gene flanked by Ptrc promoter
Lab collection



and 5ST1T2 terminator


pYA4464
Vector with p15A ori sequence and cat cassette
Lab collection


pYA4749
A GFP expression vector with a p15A ori constructed by fusing
This study



DNA segments from pYA3994 and pYA4464


pYA4337
Gene encoding PB2 inserted into pcDNA3.1(-)
This study


pYA4338
Gene encoding PB1 inserted into pcDNA3.1(-)
This study


pYA4339
Gene encoding PA inserted into pcDNA3.1(-)
This study


pYA4379
Chicken Pol I promoter (CPI) and murine Pol I terminator (MTI)
This study



cloned into pcDNA3.1(-) to create a bidirectional



vector to synthesize vRNA from CPI and mRNA from CMV promoter


pYA4383
PB2 cDNA cloned into pYA4379 to synthesize mRNA by CMV promoter and vRNA by CPI
This study


pYA4384
PB1 cDNA cloned into pYA4379 to synthesize mRNA by CMV promoter and vRNA by CPI
This study


pYA4385
PA cDNA cloned into pYA4379 to synthesize mRNA by CMV promoter and vRNA by CPI
This study


pYA4386
NP cDNA cloned into pYA4379 to synthesize mRNA by CMV promoter and vRNA by CPI
This study


pYA4387
EGFP gene cloned into pYA4379 to synthesize mRNA by CMV
This study



promoter and antisense RNA (vRNA-like) by CPI


pYA4380
CPI and MTI cloned into modified pcDNA3.1 (-) to synthesize vRNA
This study


pYA4388
HA cDNA inserted into the AarI sites in pYA4380 to synthesize vRNA by CPI
This study


pYA4389
NA cDNA cloned in pYA4380 to synthesize vRNA by CPI
This study


pYA4390
M cDNA cloned in pYA4380 to synthesize vRNA by CPI
This study


pYA4391
NS cDNA cloned in pYA4380 to synthesize vRNA by CPI
This study


pYA4392
EGFP gene cloned into pYA4380 to transcribe antisense RNA (vRNA-like) by CPI
This study


pYA4688
CPI replaced with human Pol I promoter in pYA4380 to transcribe
This study



EGFP gene into antisense RNA (vRNA-like)


pYA4519
8 influenza cDNA cassettes cloned into one plasmid to synthesize
This study



vRNAs by CPI and PB2, PB1, PA and NP mRNA/protein by CMV promoter


pYA4731
The mCherry gene cloned in between CMV and BGH-polyA terminator in pcDNA3.1(-)
This study


pYA4732
The CMV-mCherry-BGH-polyA cassette from pYA4731 inserted in the SrfI site on pYA4519
This study
















TABLE 2







Primers used in this study










Primer Name
SEQ ID
Sequence
Application














CP1
1
5′-tcggtcgcttcgcggaggtggctgg-3′
Clone chicken RNA Pol I






promoter from genomic DNA





CP2
2
5′-gtgatcgccttctccggcttttttt-3′
Clone chicken RNA Pol I





promoter from genomic DNA





PI-1
3
5′-taaaagctttctgcagaattcgccctt-3′
Amplify chicken RNA Pol I





promoter (nt −415 to −1)





PI-2
4
5′-ttaggtaccacctgctcctacagacgaac-3′
Amplify chicken RNA Pol I





promoter (nt −415 to −1)





TI-1
5
5′-taaggtaccacctgctgctcccccccaacttc-3′
Amplify murine Pol I terminator (41bp)





TI-3
6
5′-ttagctagcgtgtcgcccggagta-3′
Amplify murine Pol I terminator (41bp)





BsmBI-EGFP1
7
5′-taacgtctctctgtagtagaaacaagg
Add nontranslational sequence




tagttttttacttgtacagctcg-3′
of M segment to EGFP gene





BsmBI-EGFP2
8
5′-
Add nontranslational sequence




ttacgtctctggggagcaaaagcaggtagatattg
of M segment to EGFP gene




aaagatggtgagcaagggcg-3′





FP-cherry
9
5′-acctctagaatggtgagcaagggcgag-3′
Clone mCherry gene into pcDNA31(—)





RP-cherry
10
5′-taagaattcttacttgtacagctcgtc-3′
Clone mCherry gene into pcDNA31(—)





P1
11
5′-taactcgagatggaaagaataaaag-3′
Clone PB2 ORF into pcDNA3.1(—)





P2
12
5′-ttaggtaccctaattgatggccatc-3′
Clone PB2 ORF into pcDNA3.1(—)





P3
13
5′-taactcgagatggatgtcaatccga-3′
Clone PB1 ORF into pcDNA3.1(—)





P4
14
5′-ttaggtaccctatttttgccgtctg-3′
Clone PB1 ORF into pcDNA3.1(—)





P5
15
5′-taactcgagatggaagattttgtgc-3′
Clone PA ORF into pcDNA3.1(—)





P6
16
5′-ttaggtaccctatctcaatgcatgt-3′
Clone PA ORF into pcDNA3.1(—)





AarI-PB2-1
17
5′-taacacctgcagtcctgtagtagaaacaaggtcgt-3′
Clone PB2 cDNA into pYA4379





AarI-PB2-2
18
5′-ttacacctgcgactggggagcgaaagcaggtcaat-3′
Clone PB2 cDNA into pYA4379





AarI-PB1-1
19
5′-taacacctgcagtcctgtagtagaaacaaggcatt-3′
Clone PB1 cDNA into pYA4379





AarI-PB1-2
20
5′-ttacacctgcgactggggagcgaaagcaggcaaac-3′
Clone PB1 cDNA into pYA4379





BsmBI-PA-1
21
5′-taacgtctctctgtagtagaaacaaggtact-3′
Clone PA cDNA into pYA4379





BsmBI-PA-2
22
5′-ttacgtctctggggagcgaaagcaggtactg-3′
Clone PA cDNA into pYA4379





BsmBI-NP-1
23
5′-taacgtctctctgtagtagaaacaagggtat-3′
Clone NP cDNA into pYA4379





BsmBI-NP-2
24
5′-ttacgtctctggggagcaaaagcagggtaga-3′
Clone NP cDNA into pYA4379





BsmBI-HA-1
25
5′-taacgtctctctgtagtagaaacaagggtg-3′
Clone HA cDNA into pYA4380





BsmBI-HA-2
26
5′-ttacgtctctggggagcaaaagcaggggaa-3′
Clone HA cDNA into pYA4380





AarI-NA-1
27
5′-taacacctgcagtcctgtagtagaaacaaggagtt-3′
Clone NA cDNA into pYA4380





AarI-NA-2
28
5′-ttacacctgcgactggggagcgaaagcaggagttt-3′
Clone NA cDNA into pYA4380





BsmBI-M-1
29
5′-taacgtctctctgtagtagaaacaaggtagt-3′
Clone M cDNA into pYA4380





BsmBI-M-2
30
5′-ttacgtctctggggagcaaaagcaggtagat-3′
Clone M cDNA into pYA4380





BsmBI-NS-1
31
5′-taacgtctctctgtagtagaaacaagggtgt-3′
Clone NS cDNA into pYA4380





BsmBI-NS-2
32
5′-ttacgtctctggggagcaaaagcagggtgac-3′
Clone NS cDNA into pYA4380





SrfI-PB2
33
5′-taagcccgggcgttgacattgattattg-3′
Amplify PB2 dual promoter cassette





NgoMIV-NotI-
34
5′-ttagccggcttagcggccgccatagagcccaccgcat-3′
Amplify PB2 dual promoter cassette


PB2





BssHII-PB1
35
5′-taagcgcgcgttgacattgattattgac-3′
Amplify PB1 dual promoter cassette





NgoMIV-SbfI-
36
5′-ttagccggcttacctgcaggccatagagcccaccgca-3′
Amplify PB1 dual promoter cassette


PB1





KpnI-PA
37
5′-taaggtaccgttgacattgattattgac-3′
Amplify PA dual promoter cassette





NgoMIV-PacI-
38
5′-ttagccggcttattaattaaccatagagcccaccgca-3′
Amplify PA dual promoter cassette


PA





ApaI-NP*
39
5′-taagggcccgttgacattgattattgac-3′
Amplify NP dual promoter cassette





NgoMIV-PmII-
40
5′-ttagccggcttacacgtgccatagagcccaccgcatc-3′
Amplify NP dual promoter cassette


NP*





PmII-HA
41
5′-taacacgtggtgtcgcccggagtactgg-3′
Amplify HA mono promoter cassette





NgoMIV-HA
42
5′-ttagccggctcggtcgcttcgcggaggt-3′
Amplify HA mono promoter cassette





PacI-NA
43
5′-taattaattaagtgtcgcccggagtact-3′
Amplify NA mono promoter cassette





NgoMIV-NA
44
5′-ttagccggcttagggccctcggtcgcttcgcggag-3′
Amplify NA mono promoter cassette





SbfI-M
45
5′-taacctgcagggtgtcgcccggagtact-3′
Amplify M mono promoter cassette





NgoMIV-M
46
5′-ttagccggcttaggtacctcggtcgcttcgcggag-3′
Amplify M mono promoter cassette





NotI-NS
47
5′-taagcggccgcgtgtcgcccggagtact-3′
Amplify NS mono promoter cassette





NgoMIV-NS
48
5′-ttagccggcttagcgcgctcggtcgcttcgcggag-3′
Amplify NS mono promoter cassette





*also used to amplify CMV-mCherry-BGH cassette from pYA4731 to construct pYA4732






Cell culture. Chicken embryonic fibroblasts (CEFs) were prepared by standard trypsinization of decapitated 8-day old embryos. CEFs, human embryonic kidney (HEK293) cells and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. To co-culture CEFs and MDCK cells, each cell type was grown in 75 cm2 flasks, trypsinized, and ⅓ volume of each was mixed with growth media to a total volume of 40 ml. The mixed cells were seeded into six-well plates at 3 ml per well. All cells were maintained at 37° C. in 5% CO2.


Construction of chicken Pol I promoter-based reporter plasmids. Plasmid pcDNA3.1(−) (Invitrogen, Carlsbad, Calif.) carrying the cytomegalovirus (CMV) promoter and the bovine growth hormone (BGH) polyadenylation signal that together form the Pol II promoter-terminator system, was used to construct vector pYA4379 (SEQ ID NO:57). Briefly, chicken Pol I promoter (CPI) was cloned from chicken genomic DNA (18). The truncated murine Pol I terminator (MTI) was amplified from plasmid pTM-Pol I-WSN-All. Using unique enzyme sites introduced by PCR, CPI region (nt: −415 to −1) and MTI (41 bp) were connected with KpnI site to produce SEQ ID NO:61 (Table 3), and placed between NheI and HindIII on pcDNA3.1(−) downstream of the CMV promoter to construct the bidirectional transcription vector pYA4379 (SEQ ID NO:57) (FIG. 1A). The two AarI sites introduced inbetween CPI and MTI will allow cloning of an insert without introducing any additional nucleotides at either end. Plasmid pYA4380 (SEQ ID NO:58) was constructed by excising the CMV promoter fragment from pcDNA3.1(−) using enzymes SpeI and HindIII followed by insertion of the CPI-MTI fusion product (FIG. 1A).










TABLE 3





Sequence of fused CPI and MTI. Sequence of chicken RNA



polymerase I promoter (CPI) is underlined and sequence of


murine Pol I terminator (MTI) is given in bold. Sequence of


two Aarl sites is highlighted with gray background.

















SEQ ID NO: 61

TCGGTCGCTTCGCGGAGGTGGCTGGGGCACGGCGGAAC









GGTCTACCTGGTCCCGGCGGGCACCGTCCGGCTCGGTC








TCTCCGCGGCGGCGGCGGCTAGGGGTCGCTGCCGGGG








CGTCTCGGAAACGGCGGAACGGTCTACCCGGGTGCTAC








CGTCTCGCGCTCTCCGCGGCGGCGGCTAGAGGTCGCTG








CCGGGGCGGCTTGCGATCCGCGTCCAGGTCTACCCCGT








TTCGGATTGTCTTGGCCGCTCTGGCTGTGGGGGGGGGC








GCTACAGCTCCGGAGCTGCCAGAGGCGTCGCTGTAATTT








TGTACCTCCAGTTACGTCGAGGTAAACCTCGGCTGCCGT








CGGAGCCGCTGCCGGTAGTCGGCGCCTATGGGACTAGA








ACGTTTTTTTCGGATGCCTTATATGTTCGTCTGTAGGA








GTAC TGCTCCCCCCCAACTTCGGAGGT








CGACCAGTACTCCGGGCGACAC










Plasmid pIRES-EGFP (Clontech; Mountain View, Calif.) was the source of the enhanced green fluorescent protein (EGFP) gene used to measure promoter activities in plasmids pYA4379 (SEQ ID NO:57) and pYA4380 (SEQ ID NO:58). The EGFP gene was amplified by PCR from pIRES-EGFP using primers that introduce 5′ and 3′ non-translating sequences (NTS) from M segment of the WSN virus. The 5′-NTS-EGFP-NTS-3′ fragment was cloned into the AarI sites in-between CPI and MTI in plasmid pYA4379 (SEQ ID NO:57) and in pYA4380 (SEQ ID NO:58) to obtain plasmids pYA4387 and pYA4392, respectively (FIG. 1B). Plasmid pYA4688 was derived from pYA4392 bp replacing the chicken Pol I promoter with human Pol I promoter derived from pTM-Pol I-WSN-All (FIG. 1B). Genes encoding PB2, PB1 and PA were individually cloned into plasmid pcDNA3.1(−) to obtain plasmids pYA4337, pYA4338 and pYA4339, respectively. In transfection experiments, those three plasmids were used in combination with plasmid pCAWS-NP to provide viral polymerase and nucleoprotein.


Construction of the 8-unit plasmid pYA4519 (SEQ ID NO:60). The 8-unit plasmid pYA4519 was constructed in four stages: a) Construction of eight 1-unit plasmids. Plasmid pTM-PolI-WSN-All provides the whole set of genomic cDNAs of influenza A/WSN/33 virus. The cDNA fragments for PB2, PB1, PA, and NP were individually transferred into the AarI sites on pYA4379 (SEQ ID NO:57) to obtain plasmids pYA4383, pYA4384, pYA4385, and pYA4386, respectively (Table 1, FIG. 3). Each of the HA, NA, M and NS cDNAs was similarly cloned into pYA4380 (SEQ ID NO:58) to obtain plasmids pYA4388, pYA4389, pYA4390, and pYA4391, respectively (Table 1, FIG. 3). b) Construction of cloning vector p15A-T. DNA fragments from two different plasmids were fused to construct the cloning vector p15A-T: Plasmid pYA4464 (Table 1) was the source for p15A ori and the cat gene and plasmid pYA3994 was the source of the Ptrc-GFP-5ST1T2 expression cassette. An approximately 2550 bp DNA fragment containing both p15A-origin of replication and the cat gene was excised from plasmid pYA4464. A 1400 bp Ptrc-GFP-5ST1T2 expression cassette was amplified from plasmid pYA3994 (Table 1) using primers that introduced sites for enzymes SnaBI and AhdI towards the 5′ end and sites for AhdI and BglII towards the 3′ end of the cassette. The PCR product was digested at the ends and ligated with the previously obtained 2550 by fragment to generate a 3900 bp GFP expression vector pYA4749 (SEQ ID NO: 59, FIG. 4). The GFP expression cassette was excised out of pYA4749 bp digesting with AhdI leaving behind a linear 2530 bp vector p15A with a 3′-T overhang (generated due to AhdI digestion, FIG. 4). This linear vector will henceforth be referred to as plasmid p15A-T and will be used for convenient insertion of DNA fragments with an additional overhanging A nucleotide. c) Cloning of dual-promoter cassettes into p15A-T. cDNA cassettes of PB1, PB2, PA, and NP, along with their promoter-terminator bidirectional elements were individually amplified from pYA4384, pYA4383, pYA4385, and pYA4386, respectively, using high fidelity Pfu polymerase (PfuUltra, Stratagene) and primers that introduced unique restriction sites at both the 5′ and the 3′ ends of the PCR products. To generate a 3′-A overhang, the four amplicons were individually mixed with 5U of Taq DNA polymerase (New England Biolabs) and 0.5 mM dATP at 37° C. for 30 min. Purified products were each ligated with p15A-T linear vector to obtain four 1-unit plasmids p15A-PB2, p15A-PB1, p15A-PA, and p15A-NP (Table 1 and FIG. 5, upper panel). To construct 2-unit plasmids, mono-promoter cassettes of the remaining four viral genes (HA, NA, M, and NS) were amplified from plasmids pYA4388, pYA4389, pYA4390, and pYA4391, respectively, and cloned into unique restriction sites available on each of the 1-unit plasmids (FIG. 5). For instance, the CPI-NS-MTI fragment was amplified from pYA4391 using primers that engineer NotI and NgoMIV sites at the ends of the amplicon and was then cloned into the same sites on the 1-unit plasmid p15A-PB2 to obtain a two-unit plasmid p15A-PB2-NS (FIG. 5). Plasmids p15A-PB1-M, p15A-PA-NA, and p15A-NP-HA were also constructed by a similar procedure. As a step-wise incremental process, cDNA fragments from two different 2-unit plasmids were fused to obtain a 4-unit plasmid. The structures of the two 4-unit plasmids p15A-PB2-NS-PB1-M (12.9 kb) and p15A-PA-NA-NP-HA (13.3 kb) were shown (FIG. 5). d) Fusion of eight cDNA cassettes into a single plasmid. The DNA fragment containing PA-NA-NP-HA cassettes was excised from p15-PA-NA-NP-HA and cloned in between the KpnI and NgoMIV sites in the other four-unit plasmid to obtain the single 8-unit plasmid pYA4519 (FIG. 5) (SEQ ID NO:60). It is a 23.6 kb long plasmid containing unique restriction sites (SrfI, NotI, BssHII, SbfI, KpnI, PacI, ApaI, PmlI and NgoMIV) between every two cassettes and plasmid backbone to facilitate either any addition or replacement of genes in this plasmid. During the construction of the 4-unit and 8-unit plasmids, large DNA fragments were stained with crystal violet to avoid DNA damaging effects of ultraviolet light (30). These manipulations can also be performed in laboratory space equipped with yellow fluorescent lighting fixtures.


The 711 bp mCherry gene was amplified from pYS1190 (Table 1) and cloned between the CMV promoter and BGH terminator sequences on plasmid pcDNA3.1(−) to generate the reference plasmid pYA4731. The CMV-mCherry-BGH-polyA cassette was amplified from pYA4731 and cloned into the SrfI site on plasmid pYA4519 (SEQ ID NO:60) to obtain pYA4732 (pYA4519-mCherry) (Table 1).


Transfection. CEFs and HEK293 cells grown in 6-well plates were transfected according to the manufacturer's instructions. Briefly, 2 μl of Lipofectamine 2000 (Invitrogen) per pg plasmid DNA were individually diluted in 100 μl of Opti-MEM. After 5 min incubation at room temperature (RT), the diluted transfection reagent was mixed with the DNA. After 40 min incubation at RT, the transfection mix was added to pre-washed cells. After further incubation at RT for 3 h, the transfection medium was replaced with DMEM supplemented with 10% FBS. At 24 h post transfection, images were acquired using the Zeiss Axio Cam Mrc-5 mounted onto a Zeiss Axioskop 40-fluorescent microscope.


Virus generation. For generation of influenza virus, CEFs or co-cultured CEFs/MDCK cells were transfected with plasmid DNA as described above. After 3 h incubation, the transfection medium was replaced with 2 ml of Opti-MEM containing 0.3% bovine serum albumin (BSA), penicillin and streptomycin. At 24 hr post transfection, each well was supplemented with 1 ml of Opti-MEM containing 2 μg/ml TPCK-trypsin, 0.3% BSA, penicillin and streptomycin. At three to six days post transfection, cell supernatants were titrated onto MDCK cell monolayers to estimate influenza virus titer. All experiments were done in triplicates.


Example 1
EGFP Expression in Vectors with Dual- or Mono-Promoter Unit

The bidirectional dual promoter transcription vector pYA4379 (SEQ ID NO:57) was constructed by inserting Pol I promoter-terminator elements in plasmid pcDNA3.1(−). Here, cytomegalovirus promoter (CMV) and bovine growth hormone (BGH) polyadenylation signal (BGH) together constitute the Pol II promoter-terminator unit to synthesize mRNA, whereas, chicken Pol I promoter (CPI) and murine Pol I terminator (MTI) together constitute the Pol I promoter-terminator unit to transcribe antisense RNA of the target gene (FIG. 1A). Alternatively, the unidirectional vector pYA4380 (SEQ ID NO:58) containing the Pol unit but lacking the CMV promoter was created for the synthesis of antisense RNA alone (FIG. 1A). Plasmids pYA4387 and pYA4392 were derived from pYA4379 (SEQ ID NO:57) and pYA4380 (SEQ ID NO:58), respectively, by inserting the reporter gene EGFP between CPI and MTI to monitor the promoter activities in both plasmids (FIG. 1B). Another unidirectional plasmid pYA4688 was derived from pYA4392 by replacing human the Pol I promoter (HPI) for CPI and used as a control for monitoring EGFP synthesis (FIG. 1B).


To test the promoter activity in each plasmid, CEFs were independently transfected with plasmids (pYA4387 and pYA4392) and HEK293 cells were transfected with plasmid pYA4688 to monitor EGFP expression as a measure of promoter activity. CEFs tranfected with pYA4387 were visibly green confirming the synthesis of a functional EGFP protein (FIG. 2A). As expected, synthesis of EGFP was not observed in CEFs or in HEK293 cells when transfected with the either pYA4392 or pYA4688 (data not shown), as only the vRNA-like antisense RNA was synthesized by the Pol I unit in both cases. Expression was restored only upon co-transfection with pYA4337 (PB2), pYA4338 (PB1), pYA4339 (PA) and pCAWS-NP that together provide influenza RNA polymerases and the nucleoprotein required for vRNA replication and transcription to synthesize a functional EGFP (FIGS. 2B and C). These data suggested that pYA4387, pYA4392 and pYA4688 (and thus the parent plasmids pYA4379 (SEQ ID NO:57) and pYA4380 (SEQ ID NO:58)) carry functional promoter-terminator units and could transcribe the cloned cDNA into vRNA-like molecules in CEFs. However, the percentage of cells expressing EGFP was higher in HEK293 than in CEFs (FIG. 2).


Example 2
One-Plasmid System pYA4519 (SEQ ID NO:60)

We chose influenza A/WSN/33 virus as our model virus and cDNAs for all eight segments were obtained from the plasmid pTM-PolI-WSN-All. FIG. 5 outlines the sequential construction of plasmids to obtain the 8-unit plasmid pYA4519 (SEQ ID NO:60). To generate an 8-unit one-plasmid construct, we first constructed a p15A-T cloning vector from two plasmids pYA4464 and pYA3994 (see Materials and Methods). We amplified bidirectional cassettes of PB2, PB1, PA, and NP from plasmids pYA4383, pYA4384, pYA4385, and pYA4386, respectively (see Materials and Methods, and Table 1), and cloned individually into the p15A-T vector to obtain four 1-unit plasmids expressing viral mRNA (FIG. 5). The vRNA expression cassettes (CPI-cDNA-MTI) for HA, NA, M, and NS were then cloned into the 1-unit plasmids to obtain four 2-unit plasmids (FIG. 5). Two 2-unit plasmids were fused to obtain a 4-unit plasmid and two of those were ligated together to obtain a 23.6 kb-long 8-unit plasmid pYA4519 (SEQ ID NO:60) (FIG. 5). Plasmid pYA4519 (SEQ ID NO:60) contains a p15A origin of replication adjacent to a chloramphenicol resistance gene (cat). It is designed to synthesize both vRNA and mRNA from cDNA of each of PB1, PB2, PA and NP and vRNA from cDNA of each of HA, NA, M, and NS segments. The origin of replication, the resistance marker or any of the antigenic elements from this plasmid can be conveniently replaced with any other phenotypic determinants to generate reassortant influenza virus in cultured cells. Unique restriction sites also facilitate addition of a reporter gene cassette to monitor transfection efficiency of the plasmid. Plasmid pYA4519 (SEQ ID NO:60) was stably maintained at 37° C. in E. coli strains containing a recA mutation.


Example 3
Transfection Efficiency of pYA4519 (SEQ ID NO:60)

To determine the transfection and nuclear targeting efficiency of pYA4519 (SEQ ID NO:60), we introduced the mCherry gene into the vector pcDNA3.1(−) downstream of the CMV promoter to generate pYA4731 (pcDNA-mCherry). The entire CMV-mCherry-BGH-polyA cassette was then transferred into the 8-unit plasmid pYA4519 (SEQ ID NO:60) to generate pYA4732 (pYA4519-mCherry) and then to compare the expression of the reporter gene in CEFs and HEK293 cells. Expression of the mCherry gene from the reference plasmid pYA4731 was similar in both CEFs and HEK293 cells (FIGS. 6A and E), suggesting similar levels of transfection and nuclear translocation efficiency of the small plasmid in both cell lines. However, CEFs and HEK293 cells differed in both aspects when synthesis of mCherry from the large plasmid pYA4732 was compared (FIGS. 6B and F). The level of mCherry synthesis from pYA4732 was much higher in HEK293 (FIG. 6E) than in CEFs (FIG. 6B). Expression of mCherry from pYA4732 was comparable to that from the reference plasmid pYA4731 in case of HEK293 cells (FIGS. 6E and F), whereas, in CEFs the efficiency decreased dramatically with the increase in plasmid size (compare FIGS. 6A and B). We hypothesized that the lower mCherry synthesis in CEFs (from pYA4732) may be due to limited translocation of the large plasmid into the CEFs nucleus. To test this hypothesis, we co-transfected CEFs with pYA4732 and pYA4392 (pYA4380-EGFP) and co-transfected HEK293 with pYA4732 and pYA4688 to measure the synthesis of both mCherry (FIGS. 6C and G) and EGFP (4D and F) proteins from the same field. Since the EGFP gene is cloned between the CPI-MTI Pol I unit on pYA4392 and the HPI-MTI Pol I unit on pYA4688 (resulting only in the generation of vRNA-like molecules), synthesis of a functional EGFP protein in either case is only possible in the presence of all the viral polymerases and the nucleoprotein provided from plasmid pYA4732. We observed EGFP synthesis both in HEK293 and CEFs, but the percentage of HEK293 cells synthesizing both mCherry and EGFP was much greater than in the CEFs (compare FIGS. 6C and D with FIGS. 6G and F) suggesting a lower translocation of the 8-unit plasmid into the CEFs nucleus.


Example 4
Generation of Influenza Virus from Plasmid(s)

Efficiency of influenza virus recovery was compared between our 1-unit eight-plasmid system (plasmids pYA4383, pYA4384, pYA4385, pYA4386, pYA4388, pYA4389, pYA4390, and pYA4391) and our novel one-plasmid 8-unit system pYA4519 (SEQ ID NO:60). Cultured CEFs were either transfected with pYA4519 (SEQ ID NO:60) or co-transfected with eight plasmids (pYA4383, pYA4384, pYA4385, pYA4386, pYA4388, pYA4389, pYA4390, and pYA4391) to provide all the necessary viral components as described in Materials and Methods. The mean titer at 3-days and 6-days post transfection was approximately 300 and 1×103 PFU/ml influenza viruses, respectively, when transfected with pYA4519 (SEQ ID NO:60), whereas the virus yield using the eight-plasmid method estimated at the same time points was approximately 50 and 700 PFU/ml, respectively, (Table 4). Virus yield was much higher in cocultured CEFs/MDCK cells transfected by plasmid pYA4519 (SEQ ID NO:60) with approximately 1×104 PFU/ml and 1×108 PFU/ml estimated on the 3 and 6 days post transfection, respectively. This was expected as MDCK cells are known to support the growth of influenza virus better than CEF cells. Together these results suggested that recovery of influenza virus from pYA4519 (SEQ ID NO:60) transfected cells was more efficient than from the previously developed eight-plasmid system.









TABLE 4







Influenza A virus generation in CEFs (PFU/ml)










3rd day post transfection
6th day post transfection













Plasmid(s)
No. 1a
No. 2a
No. 3a
No. 1a
No. 2a
No. 3a
















8 × 1-unit
40
60
60
1280
440
480


plasmidsb


PYA4519
400
260
280
1800
1000
1000






aTriplicate wells.




bPlasmids pYA4383, pYA4384, pYA4385, pYA4386, pYA4388, pYA4389, pYA4390, and PYA4391.







Discussion for Examples 1-4

The goal of this study was to construct the influenza virus genome on a single plasmid and rescue the virus from cultured chicken cells. We chose the influenza virus WSN strain as the model virus and with the combination of reverse genetics and the dual promoter system successfully constructed an 8-unit plasmid pYA4519 (SEQ ID NO:60). Care was also taken to limit the use of multiple CMV promoters in our plasmid to reduce the number of repetitive sequences that may promote intra-plasmid recombination and thus decrease plasmid stability. The 8-unit plasmid was designed to produce influenza polymerase complex (PB1, PB2 and PA), nucleoprotein (NP) and 8 viral RNAs (PB1, PB2, PA, NP, HA, NA, M and NS) in avian cells (FIG. 5). By transfection, the “one-plasmid” system showed more efficient virus generation in CEFs than our 1-unit (a unit stands for a cDNA corresponding to one influenza segment, it may be flanked only by Pol I and MTI, or flanked by both Pol I/Pol II plus their terminators) eight-plasmid system (Table 4). Generation of influenza virus from a minimal number of plasmid constructs has been a long-term challenge and through this study for the first time we demonstrated successful recovery of influenza virus from expression of a single plasmid.


Factors such as plasmid constructs used, and the host cell line, affect the efficiency of virus recovery (22), and our study provides additional vital evidence in their support. We compared both transfection and viral recovery efficiency between CEFs and HEK293 cells. Both cell types could be transfected with equal efficiency when smaller size plasmids were used (FIG. 2 and FIGS. 6A and E). The viral yield however was higher in HEK293 cells when compared to CEFs. This difference could be attributed to either lower production of vRNAs, or lower conversion from vRNAs to protein or both, in chicken cells. Transfection experiments involving the large size plasmid pYA4519-mCherry (25.3 kb), however, indicated that HEK293 cells are better recipients than the CEFs. Two important conclusions can be drawn from these observations; firstly, our data suggested that plasmid size plays an important role in successful viral recovery. Whereas efficient virus recovery and reporter gene expression in CEFs was possible by transfecting with multiple smaller plasmids (FIG. 2 and Table 4), a similar attempt using a larger plasmid (25.3 kb) had limited success, suggesting the plasmid size as a potential limiting factor. Alternatively, expression might improve in other avian species or in different cells in those species. Secondly, it is known that virus recovery is higher in 293T cells than in Vero cells or CEFs (18, 22, 23, 27). This was one important criterion for higher virus yield from the three-plasmid system developed by Neumann et. al. Our results indicated that HEK293 cells are not only highly transfectable cells, but also can be transfected with large size plasmids. Furthermore, certain cell-specific factors in HEK293 cells seem to promote nuclear translocation of larger plasmids more effectively than other cell types such as CEFs. We are hence working towards improving translocation of pYA4519 (SEQ ID NO:60) into the nucleus of CEFs by including a nuclear targeting sequence, such as the promoter/enhancer region of simian virus 40 (SV40) (6).


Our plasmid construct should also facilitate the design of a much simpler approach to develop influenza vaccine seeds. Currently, influenza vaccine seeds use the “2+6” strategy, in which the HA and NA segments are taken from an epidemic strain and the remaining 6 segments of the influenza viral genome are taken from either the high productive strain PR8 (A/PR/8/34) or the cold-adapted strain (e.g. A/AA/6/60) (4, 10, 12). Construction of one plasmid producing all the necessary backbone segments and proteins from donor virus provides a simpler and more efficient “1+2” approach to generate influenza vaccine seeds.


The currently used influenza vaccines for human use are the inactivated and attenuated forms of the virus and are administered via the intramuscular or the intranasal routes. Manufacturing these vaccines using cell culture or embryonated chicken eggs is both expensive and a time-consuming process. An inexpensive and oral influenza vaccine remains a medical priority, especially for pandemic influenza. Our one plasmid offers a viable option to generate attenuated influenza virus in vivo where the plasmid can be delivered orally or intranasally using a recombinant bacterial strain. Our laboratory has been successful in constructing recombinant attenuated strains of Salmonella enterica Serovar Typhimurium that are designed for enhanced antigen delivery in the host and ensure regulated delayed lysis of the pathogen to inhibit long-term host colonization (5). To construct such an attenuated strain that could effectively deliver plasmid DNA into the host will be the next step towards developing a recombinant bacteria based-vaccine against influenza to be used both in the poultry industry and for pandemic influenza.


In our pilot study, we choose the influenza virus WSN strain for validation of our one-plasmid system. For developing a bacterial based influenza vaccine, the expression vector must be modified to generate attenuated influenza virus. One strategy would be constructing the single expression vector with HA and NA from epidemic influenza virus and the other 6 segments from a cold-adapted influenza strain (e.g. A/AA/6/60) (4, 12). Another strategy is to introduce mutations into viral polymerase coding genes and another to employ a truncated NS1 (nonstructural protein 1) gene to obtain attenuated influenza virus (7, 29, 33). Additionally, the HA segment from influenza vaccine may form a new ressortant virus with the other segments from a preexisting influenza virus in the host. The polybasic cleavage peptides of the HA proteins are required for high pathogenicity of influenza viruses (36). Thus, for vaccine development, the polybasic cleavage site in HA will be replaced with a consensus sequence derived from HA-encoding sequences from avirulent strains (28, 33).


Example 5
Construction of the 8-Unit Plasmid pYA4562

Optimal gene expression from the 8-unit plasmid requires efficient translocation of the plasmid construct into the nucleus of the host cells. Nuclear targeting sequence and NF-κB binding site have been reported to improve the nuclear import of DNA construct (6, 19). In our study, transfection of chicken cells with plasmid pYA4732 did not result in efficient expression of mCherry (Example 3). One possible reason is the lack of a nuclear targeting sequence to facilitate the nuclear import of pYA4732 (and its parental plasmid pYA4519). Here the SV40 nuclear targeting sequence (SV40 DTS) and NF-κB binding site were introduced into plasmid pYA4519 to enhance its nuclear import. The SV40 DTS was obtained from a commercial vector pBICEP-CMV-3 (Sigma) and the NF-κB binding site was obtained from plasmid pYA4545 (from Clonebank in Curtiss' lab). Then they were fused with a kanamycin-resistance cassette (kan) by PCR. The entire fusion fragment was inserted into the SrfI site of pYA4519 to generate pYA4562 (FIG. 7). This modification has led to higher virus yield in bacterial carrier-mediated plasmid delivery (example 9).


Example 6

Salmonella Mediated Delivery of EGFP Reporter Plasmid pYA4336

To mediate the delivery of plasmid DNA, an auxotrophic Salmonella Typhimurium strain χ9052 (ΔasdA33 Δalr-3 ΔdadB4) was selected. Inactivation of the asd gene causes an obligate requirement for the essential amino acid diaminopimelic acid (DAP), whereas inactivation of both the alr and dadB genes confers an absolute requirement for D-alanine. Both DAP and D-alanine are essential unique subunits of the peptidoglycan ridgid layer of the bacterial cell wall. A replicating bacterial cell requires these components for cell wall synthesis and neither of these amino acids is present in animal tissues. In the absence of these nutrients in the host cell, the integrity of the bacterial cell wall is compromised and the bacterium undergoes lysis in the host. Lysis of the intracellular bacterial cell would release the expression vector into the host cytoplasm, and the nuclear targeting sequence(s) on the vector would then promote the translocation of the expression vector into the nucleus, ultimately resulting in the desired expression of viral genes. The conditional growth on LB agar plates with or without supplement(s) was observed for three bacterial carriers, including χ8276 (ΔasdA27), χ8901 (Δalr-3 ΔdadB4) and χ9052 (ΔasdA33 Δalr-3 ΔdadB4). The wild-type S. Typhimurium control strain showed growth on each plate (FIG. 8A). In another assay, each Salmonella carrier was resuspended and incubated in LB broth without any supplements for 24 hours. Then the bacterial cells were gently pelleted (8,000 rpm for 5 min) and stained with Live/Dead BacLight Bacterial Viability kit (Molecular probes, cat. L13152). Under the fluorescence microscope the carrier strains χ8276, χ8901 and χ9052 showed much bigger size and more dead cells (red fluorescence) than the wild-type strain χ3761 (FIG. 8B). Surprisingly, the genomic DNA stained with PI (red fluorescence) was even found to flush out from the dead bacterial cells. Comparing with wild-type control, the three carrier strains showed much more cell debris that can not be stained with fluoresceins due to the loss of genomic DNA. Those data proved that the incomplete bacterial cell wall was unable to protect the bacterial cell membrane from damage of stress, permeation pressure and other factors. Plasmid pYA4336 is a derivative of pcDNA3.1(−) obtained by cloning the EGFP gene under the control of the CMV promoter (FIG. 8C). Salmonella Typhimurium χ8276, χ8901 and χ9052 carrying pYA4336 each was cultured in 3 ml of LB medium containing 100 μg/ml DL-alanine, 50 μg/ml diaminopimelic acid (DAP) and 100 μg/ml ampicillin at 30° C. The bacterial pellet was resuspended in DMEM without fetal bovine serum (FBS) and antibiotics. Chicken embryonic fibroblasts (CEFs) cultured in a 6-well plate were incubated with the bacteria at 37° C. for 1 h. 24 hours later, the cells were observed in the fluorescence microscope for EGFP expression (FIG. 8D). Though EGFP expressing cells could be observed from CEFs infected by either of the bacterial carriers, the χ9052(pYA4336) seemed to result in the most efficient plasmid delivery in repeated experiments (data not shown).


Example 7
Determination of the Structural Integrity of the 8-Unit Plasmid in Strains of Salmonella Typhimurium

For bacterial carrier-mediated plasmid delivery, it is essential that the structural integrity of the target plasmid construct be maintained. RecA and RecF (encoded by genes recA and recF, respectively) catalyze recombination of homologus DNA sequences on one plasmid or between two plasmids. The 8-unit plasmid construct carries numerous such repeated DNA elements in the form of Pol I and Pol II promoters and terminators. These repeated sequences are very good substrates for both RecA- and RecF-enzyme mediated recombination. We hence determined the individual effect of the inactivation of these genes in Salmonella.


The recA and recF deletion mutations were individually introduced into Salmonella Typhimurium χ9052 (ΔasdA33 Δalr-3 ΔdadB4). The resulting strains are χ9834 (ΔasdA33 Δalr-3 ΔdadB4 ΔrecA62) and χ11018 (Δasd-33 Δalr-3 ΔdadB4 ΔrecF126), respectively.



Salmonella strains χ9052, χ9834 and χ11018 were each transformed with plasmid pYA4519, plated onto LB plates and incubated overnight at 37° C. From each strain, a correct clone was obtained and diluted 1:1000 into 3 ml LB medium and grown at 37° C. for 12 h. The dilution and growth process was repeated for 4 additional cycles. Plasmid DNA was extracted from 1.5 ml of culture from each cycle of growth. An aliquot of plasmid from each sample was digested with BamHI and separated on a 1.2% agarose gel. Bacteria from the final cultures were spread onto supplemented LB-agar plates and incubated overnight at 37° C. Plasmid DNA was extracted from single colonies and structural integrity of the plasmid was verified by comparing the restriction profile upon BamHI digestion (FIG. 9). Accumulated recombination events lead to gene deletions on plasmid pYA4519, therefore resulting in changes of the restriction map generated by BamHI digestion.


We noted that at time 0, before passage, the plasmid yield from the Rec+ strain, χ9052, was less than that obtained from the two rec mutants. After the second cycle of growth there was a reduction in the amount of DNA in most of the expected bands, indicating that the plasmid structure was deteriorating after each passage. Qualitatively, the plasmid structure appeared to be stable for the first four passages in strains χ9834 (ΔrecA62), and χ11018 (ΔrecF126). In this experiment we demonstrate that deletion of recA and recF in Salmonella Typhimurium significantly minimizes Rec-dependent recombination of the plasmid, thus ensuring structural integrity of our 8-unit plasmid in spite of repetitive sequences.


Example 8
χ9834-Mediated Delivery of Plasmid pYA4732

The goal of this experiment was to determine whether Salmonella could mediate the delivery of the large expression vector into cultured chicken cells. Plasmid pYA4732 (FIG. 10A) was derived from the 8-unit plasmid pYA4519 by inserting a eukaryotic mCherry expression cassette that is from plasmid pYA4731. The mCherry gene is used as a reporter gene in this experiment; wherein, expression of the gene product signifies successful lysis of the bacterium in the host cytoplasm and eventual translocation of the plasmid construct to the host cell nucleus. Salmonella Typhimurium strain χ9834 (ΔasdA33 Δalr-3 ΔdadB4 ΔrecA62) was selected to deliver pYA4732. This strain has obligate requirements for diaminopimelic acid (DAP) and D-alanine by virtue of the ΔasdA33 Δalr-3 ΔdadB4 mutations. The strain will thus undergo lysis in the host cells in the absence of the above mentioned nutrients. Bacterial cell lysis ensures release of the plasmid DNA into the Salmonella containing vacuole (SCV) and it can then be transported into the nucleus through a yet unknown mechanism, resulting in expressing the genes under question. This strain also carries a recA62 deletion to reduce plasmid recombination in pYA4732.



Salmonella Typhimurium χ9834 carrying pYA4732 was cultured in 3 ml of LB medium containing 100 μg/ml DL-alanine, 50 μg/ml DAP and 25 μg/ml chloramphenicol at 30° C. As a control, the χ9834 carrying pYA4731 was cultured in 3 ml of LB medium containing 100 μg/ml DL-alanine, 50 μg/ml DAP and 100 μg/ml carbencillin at 30° C. The overnight cultures were pelleted and resuspended in DMEM without fetal bovine serum and antibiotics. Chicken embryonic fibroblasts (CEFs) in 6-well plates were incubated with the bacteria at 37° C. for 1 h. 24 h later, the cells were observed under fluorescence microscope. The results showed that the large plasmid pYA4732 could be delivered into cultured chicken fibroblasts and was expressed. In contrast, the small reporter plasmid pYA4731 was more efficiently delivered by the Salmonella carrier (FIG. 10B). These results suggest that the large size plasmid suffers from inefficient nuclear import in bacterial-mediated plasmid delivery as well as in transfection. Of note, the plasmid pYA4731 also expresses mCherry in prokaryotic cells, as observed in E. coli and Salmonella strains. It is most likely results from the inframe ATG codon close to the 5′ terminus and the adjacent upstream SD sequence. Therefore, live bacterial cells are observed as red spots for cells infected by χ9834(pYA4731).


Example 9
Influenza Virus Rescued from Co-Cultured CEFs/MDCK Cells by Infection with χ9834 carrying pYA4519 or pYA4562

The goal of this experiment was to determine whether Salmonella-mediated delivery of the 8-unit plasmid into chicken cells leads to the generation of influenza virus. Based on the transfection data (Table 4), the chicken embryonic fibroblasts did not support the replication of the influenza virus WSN strain (no substantial increase of virus titers between the 3rd and 6th day post transfection). The MDCK cells on the other hand are known to support the growth of the influenza virus WSN strain. A co-culture of chicken embryonic fibroblasts (CEFs) and Madin-Darby canine kidney (MDCK) cells supports the propagation of the influenza virus. Virus generated and released from transfected CEFs can infect the adjacent MDCK cells that support replication of the virus. Transfection of co-cultured CEFs/MDCK cells with the 8-unit plasmid pYA4519 resulted in higher titers of influenza virus (Example 4). Salmonella Typhimurium χ9834 carrying pYA4519 or pYA4562 were cultured in 3 ml of LB medium containing 100 μg/ml DL-alanine, 50 μg/ml DAP and 25 μg/ml chloramphenicol at 30° C. with shaking (200 rpm) for 20 h. In each case, 1 ml of bacterial culture was harvested and resuspended in 1 ml of DMEM without fetal bovine serum (FBS) and antibiotics.


CEFs and MDCK cells grown in 75 cm2 flasks were trypsinized, and ⅓ volume of each was mixed with DMEM containing 10% FBS to a total volume of 40 ml. The mixed cells were seeded into six-well plates at 3 ml per well. All cells were maintained at 37° C. in 5% CO2. The cells were washed with DPBS for three times. 100 μl, 200 μl and 500 μl of resuspended bacteria were added into each well. DMEM was added to a final volume of 1 ml and mixed by rocking back and forth. The cells were incubated at 37° C. in a CO2 incubator for 1 h. For each well, media was changed to 2 ml of Opti-MEM containing 0.3% BSA, 10 μg/ml gentamycin. One day post-infection, each well was supplemented with 1 ml of Opti-MEM containing 0.3% BSA, 10 μg/ml gentamycin and 2 μg/ml TPCK-trypsin (The final concentration is 0.7 μg/ml). Six days post-infection, supernatants from each well were collected for hemagglutination tests (Table 5) and TCID50 determinations (FIG. 11). The latter result indicates generation of active influenza virus.


CEFs/MDCK co-culture infected with χ9834 carrying pYA4562 generated higher titers of influenza virus, supporting our hypothesis that inclusion of additional nuclear targeting sequences in the 8-unit plasmid enhances the nuclear translocation, hence the viral yield.









TABLE 5







Hemagglutination test on the supernatants from


co-cultured CEFs/MDCK cells infected by Salmonella


delivering 8-unit expression plasmids











χ9834(pYA4562)
χ9834(pYA4519)
















100
200
500
100
200
500
WSN virus


Dilution
μl
μl
μl
μl
μl
μl
(Positive control)





1:2
+
+
+


+
+


1:4
+
+
+



+


1:8
+

+



+


1:16






+


1:32









1:64












+, Hemagglutination of chicken red blood cells.


−, No hemagglutination observed.






Example 10
Construction of 8-Unit Plasmids Carrying HA and NA Genes from LPAI Virus

To generate of attenuated influenza virus in vivo and to determine the immune response against the attenuated strain, it is necessary to construct a plasmid encoding an attenuated virus. So that the virus generated in vivo can be determined by virus shielding, and the immune response can be determined by subsequent challenge with influenza virus.


The influenza A virus (A/chicken/TX/167280-4/02(H5N3) is an isolate from White Leghorns chickens. It belongs to a low pathogenic avian influenza virus and causes clinical symptoms such as wheezing and swollen heads. The viral HA segment (AY296085, henceforth referred to as Tx02HA), shares homology with low pathogenic virus (16). It hence makes an ideal challenge strain. On the other hand, an avirulent influenza A virus can be generated from a single expression vector encoding Tx02HA and Tx02NA (NA segments derived from Tx02 virus) segments and the remaining 6 segments from a mouse adapted influenza virus, such as the WSN virus.


Based on these considerations, the Tx02HA and Tx02NA genes were amplified from influenza A virus (A/chicken/TX/167280-4/02(H5N3) by RT-PCR and cloned between CPI and MTI in the p15A ori plasmids pYA4591 and pYA4592 to generate plasmids pYA4593 and pYA4592-Tx02NA. The CPI-Tx02HA-MTI cassette was amplified from pYA4593 to replace the WSN HA cassette in pYA4519 to obtain plasmid pYA4693. The CPI-Tx02NA-MTI cassette was amplified from pYA4592-Tx02NA to replace the WSN NA cassette in pYA4693 to obtain plasmid pYA4929 (FIG. 12A). Subsequently, the cat and kan markers in pYA4929 were replaced with aroA cassette derived from pYA4784 which is p15A ori based AroA+ vector. The resulting plasmid was designated as pAY4930 (FIG. 12B). Both pYA4929 and pYA4930 were designed to yield an avian influenza virus of low pathogenicity suitable for immunization of poultry. In other applications, the sequence encoding the influenza virus could be modified to attenuate the strain's ability to cause disease symptoms without eliminating or adversely altering its immunogenicity, such that the immunized bird (animal) develops protective immunity against influenza virus.


Another feasible alternative is to directly inject this plasmid construct into the target host using a gene gun to also result in the generation of live attenuated influenza virus, which can also stimulate a protective immune response against other related pathogenic strains of influenza virus.


One can also vaccinate in ovo either by directly injecting the plasmid DNA into the embryonated chicken eggs or by bacterial carrier-mediated delivery to generate live attenuated influenza vaccine. Viral yield by direct injection of the plasmid DNA is at least 1000-fold lower than that obtained by delivering the plasmid construct via a bacterial carrier.


Example 11
Ongoing Studies

Our laboratory has earlier constructed a “lysis-vector” pYA3681 (FIG. 13) for the regulated delayed lysis system (15). This vector can be used in conjunction with any Salmonella strain containing asd and ΔPmurA::TT araC PBAD murA mutations, as seen in both strain genotypes described below. Three different derivatives of pYA3681 have been constructed by replacing the origin of replication: pSC101 ori (pYA4595, FIG. 13B), p15A ori (pYA4589, FIG. 13C), and pUC ori (pYA4594, FIG. 13D). Each of these plasmids can complement the ΔasdA27::TT araC PBAD c2 and ΔPmurA25::TT araC PBAD murA mutations in a Salmonella strain to form a regulated delayed lysis in vivo system. For example, a Salmonella strain carrying such a plasmid can be cultured in LB medium supplemented with 0.2% arabinose, and behaves as a wild-type strain in terms of colonization and invasion of the host. The ΔaraBAD23 mutation in turn compromises the ability of the bacterium to metabolize arabinose. Replication of bacteria in the absence of arabinose (conditions encountered in vivo) causes cessation in synthesis of Asd and MurA enzymes, which are continuously diluted at each cell division. This ultimately results in lysis of the strain and release of the bacterial cell contents, including the plasmid expression vector DNA, into the host cell cytoplasm. Compared to the direct lysis system (Examples 6, 8 and 9), the regulated delayed lysis in vivo system can improve Salmonella-mediated plasmid delivery in vivo. The plasmids with different copy numbers allow one to pre-select the timing (number of cell divisions) for Salmonella cells to begin lysing after animal inoculation/immunization.


Vaccine strain: We have generated various Salmonella Typhimurium strains listed below. We are proposing to introduce ΔrecA62 or ΔrecF126 into some strains to enhance stable maintenance of the expression vector. In other cases, we need to add ΔsifA26 or ΔendA2311 to enable escape from the endosome or prevent endonuclease cutting of released plasmid DNA, respectively. In other cases, the ΔaroA21426 mutation is added to maintain the single 8-unit plasmid specifying synthesis and assembly of influenza virus.

    • χ11017: ΔasdA27::TT araC PBAD c2 ΔaraBAD23 Δ(gmd-fcl)-26 Δpmi-2426 ΔrelA198::TT araC PBAD lacI TT ΔPmurA25::TT araC PBAD murA
    • χ11020: ΔasdA27::TT araC PBAD c2 ΔaraBAD23 Δ(gmd-fcl)-26 Δpmi-2426 ΔrelA198::TT araC PBAD lacI TT ΔPmurA25::TT araC PBAD murA ΔaroA21319
    • χ11228: ΔasdA27::TT araC PBAD c2 ΔPmurA25::TT araC PBAD murA ΔaraBAD23 Δ (gmd-fcl)-26 ΔrelA198::araC PBAD lacI TTΔpmi-2426 ΔtlpA181 ΔsseL116
    • χ11326: ΔasdA27::TT araC PBAD c2 ΔPmurA25::TT araC PBAD murA ΔaraBAD23 Δ(gmd-fcl)-26 ΔrelA198::araC PBAD lacI TTΔpmi-2426 ΔtlpA181 ΔsseL116 ΔsifA26
    • χ11327: ΔasdA27::TT araC PBAD c2 ΔPmurA25::TT araC PBAD murA ΔaraBAD23 Δ(gmd-fcl)-26 ΔrelA198::araC PBAD lacI TTΔpmi-2426 ΔtlpA181 ΔsseL116 ΔPhilA::Ptrc ΔlacO888 hilA ΔsifA26
    • χ11233: ΔasdA27::TT araC PBAD c2 ΔPmurA25::TT araC PBAD murA Δ(araC PBAD)-5::P22 PR araBAD Δ(gmd-fcl)-26 ΔrelA198::araC PBAD lacI TT Δpmi-2426 ΔaroA21419 ΔPhilA::Ptrc ΔlacO888 hilA


Vaccine vector: We have constructed a 8-unit plasmid pYA4930 with a wild-type aroA cassette (FIG. 12). This will serve two purposes: a) complementation of the ΔaroA21419 mutation in χ11020, and b) stable maintenance of pYA4930 in χ11020. AroA is an essential enzyme for the synthesis of various aromatic amino acids and vitamins, hence survival of the Salmonella strain with an ΔaroA mutation requires aminoacid and/or vitamin supplements in the growth medium. Alternatively, the mutation can be complemented by providing the gene on a plasmid. Here we chose to clone the aroA cassette in the 8-unit plasmid pYA4693, so that, the obligate requirement of the AroA enzyme (in the absence of external aromatic acid supplementation) would ensure stable maintenance of the expression vector in the strain χ11020. Additionally, we have truncated the NS1 gene which could be included in plasmid pYA4930 to attenuate the virus if necessary. Although the likelihood of this plasmid to produce a high pathogenic influenza virus is minimal (see Example 10).


The χ11020-derived strain with recA deletion (or recF deletion) will be harbored with plasmid pYA4930 and one of the lysis vectors (pYA3681, pYA4589, pYA4595, or pYA4594), so that the regulated lysis of the bacterial carrier will mediate the delivery of plasmid pYA4930.


Vaccination: Chickens will be vaccinated with the above described recombinant strains via three different routes; intranasally, orally, or intramuscularly. The influenza A virus (A/chicken/TX/167280-4/02(H5N3)) is an isolate from White Leghorn chickens. It causes clinical signs, such as wheezing and swollen heads, and belongs to a low pathogenic avian influenza virus (16). This virus will be used to challenge the immunized chickens to evaluate the protection efficiency (clinical symptoms and virus shielding).


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Sequences of this Study.


Influenza A Virus Genes.

All influenza A/WSN/33 virus genes were derived from plasmid pTM-PolI-WSN-All (A gift from Dr. Yoshihiro Kawaoka, University of Wisconsin—Madison). The sequence of each gene was listed as following.










>PB2 (SEQ ID NO: 49)










1
agcgaaagca ggtcaattat attcaatatg gaaagaataa aagaactaag




gaatctaatg





61
tcgcagtctc gcactcgcga gatactcaca aaaaccaccg tggaccatat



ggccataatc





121
aagaagtaca catcaggaag acaggagaag aacccagcac ttaggatgaa



atggatgatg





181
gcaatgaaat atccaattac agcagacaag aggataacgg aaatgattcc



tgagagaaat





241
gagcagggac aaactttatg gagtaaaatg aatgacgccg gatcagaccg



agtgatggta





301
tcacctctgg ctgtgacatg gtggaatagg aatggaccag tgacaagtac



agttcattat





361
ccaaaaatct acaaaactta ttttgaaaaa gtcgaaaggt taaaacatgg



aacctttggc





421
cctgtccatt ttagaaacca agtcaaaata cgtcgaagag ttgacataaa



tcctggtcat





481
gcagatctca gtgccaaaga ggcacaggat gtaatcatgg aagttgtttt



ccctaacgaa





541
gtgggagcca ggatactaac atcggaatcg caactaacga caaccaaaga



gaagaaagaa





601
gaactccagg gttgcaaaat ttctcctctg atggtggcat acatgttgga



gagagaactg





661
gtccgcaaaa cgagattcct cccagtggct ggtggaacaa gcagtgtgta



cattgaagtg





721
ttgcatttga cccaaggaac atgctgggaa cagatgtaca ctccaggagg



ggaggcgagg





781
aatgatgatg ttgatcaaag cttaattatt gctgctagaa acatagtaag



aagagccaca





841
gtatcagcag atccactagc atctttattg gagatgtgcc acagcacgca



gattggtgga





901
ataaggatgg taaacatcct taggcagaac ccaacagaag agcaagccgt



ggatatttgc





961
aaggctgcaa tgggactgag aattagctca tccttcagtt ttggtggatt



cacatttaag





1021
agaacaagcg gatcatcagt caagagagag gaagaggtgc ttacgggcaa



tcttcagaca





1081
ttgaagataa gagtacatga gggatatgaa gagttcacaa tggttgggag



aagagcaaca





1141
gctatactca gaaaagcaac caggagattg attcagctga tagtgagtgg



gagagacgaa





1201
cagtcgattg ccgaagcaat aattgtggcc atggtatttt cacaagagga



ttgtatgata





1261
aaagcagtta gaggtgacct gaatttcgtc aatagggcga atcagcgatt



gaatcccatg





1321
caccaacttt tgagacattt tcagaaggat gcaaaggtgc tctttcaaaa



ttggggaatt





1381
gaatccatcg acaatgtgat gggaatgatc gggatattgc ccgacatgac



tccaagcacc





1441
gagatgtcaa tgagaggagt gagaatcagc aaaatggggg tagatgagta



ttccagcgcg





1501
gagaagatag tggtgagcat tgaccgtttt ttgagagtta gggaccaacg



tgggaatgta





1561
ctactgtctc ccgaggagat cagtgaaaca cagggaacag agaaactgac



aataacttac





1621
tcatcgtcaa tgatgtggga gattaatggt cctgaatcag tgttggtcaa



tacctatcag





1681
tggatcatca gaaactggga aactgttaaa attcagtggt cccagaatcc



tacaatgctg





1741
tacaataaaa tggaatttga gccatttcag tctttagttc caaaggccgt



tagaggccaa





1801
tacagtgggt ttgtgagaac tctgttccaa caaatgaggg atgtgcttgg



gacatttgat





1861
accgctcaga taataaaact tcttcccttc gcagccgctc caccaaagca



aagtagaacg





1921
cagttctcct cattgactat aaatgtgagg ggatcaggaa tgagaatact



tgtaaggggc





1981
aattctccag tattcaacta caacaagacc actaaaagac tcacagttct



cggaaaggat





2041
gctggccctt taactgaaga cccagatgaa ggcacagctg gagttgagtc



cgcagttctg





2101
agaggattcc tcattctggg caaagaagac aggagatatg gaccagcatt



aagcataaat





2161
gaactgagca accttgcgaa aggagagaag gctaatgtgc taattgggca



aggagacgtg





2221
gtgttggtaa tgaaacggaa acggaactct agcatactta ctgacagcca



gacagcgacc





2281
aaaagaattc ggatggccat caattagtgt cgaatagttt aaaaacgacc



ttgtttctac





2341
t











>PB1 (SEQ ID NO: 50)










1
agcgaaagca ggcaaaccat ttgaatggat gtcaatccga ctttactttt




cttaaaagtg





61
ccagcacaaa atgctataag cacaactttc ccttatactg gagaccctcc



ttacagccat





121
gggacaggaa caggatacac catggatact gtcaacagga cacatcagta



ctcagaaagg





181
ggaagatgga caacaaacac cgaaactgga gcaccgcaac tcaacccgat



tgatgggcca





241
ctgccagaag acaatgaacc aagtggttat gcccaaacag attgtgtatt



ggaagcaatg





301
gccttccttg aggaatccca tcctggtatc tttgagacct cgtgtcttga



aacgatggag





361
gttgttcagc aaacacgagt ggacaagctg acacaaggcc gacagaccta



tgactggact





421
ctaaatagga accagcctgc tgcaacagca ttggccaaca caatagaagt



gttcagatca





481
aatggcctca cggccaatga atctggaagg ctcatagact tccttaagga



tgtaatggag





541
tcaatgaaca aagaagaaat ggagatcaca actcattttc agagaaagag



acgagtgaga





601
gacaatatga ctaagaaaat ggtgacacag agaacaatag gtaaaaggaa



gcagagattg





661
aacaaaagga gttatctaat tagggcatta accctgaaca caatgaccaa



agatgctgag





721
agagggaagc taaaacggag agcaattgca accccaggga tgcaaataag



ggggtttgta





781
tactttgttg agacactagc aaggagtata tgtgagaaac ttgaacaatc



aggattgcca





841
gttggaggca atgagaagaa agcaaagttg gcaaatgttg taaggaagat



gatgaccaat





901
tctcaggaca ctgaaatttc tttcaccatc actggagata acaccaaatg



gaacgaaaat





961
cagaaccctc ggatgttttt ggccatgatc acatatataa ccagaaatca



gcccgaatgg





1021
ttcagaaatg ttctaagtat tgctccaata atgttctcaa acaaaatggc



gagactggga





1081
aaggggtaca tgtttgagag caagagtatg aaaattagaa ctcaaatacc



tgcagaaatg





1141
ctagcaagca tcgatttgaa atacttcaat gattcaacta gaaagaagat



tgaaaaaatc





1201
cggccgctct taatagatgg gactgcatca ttgagccctg gaatgatgat



gggcatgttc





1261
aatatgttaa gtactgtatt aggcgtctcc atcctgaatc ttggacaaaa



gagacacacc





1321
aagactactt actggtggga tggtcttcaa tcttctgatg attttgctct



gattgtgaat





1381
gcacccaatc atgaagggat tcaagccgga gtcaacaggt tttatcgaac



ctgtaagcta





1441
cttggaatta atatgagcaa gaaaaagtct tacataaaca gaacaggtac



atttgaattc





1501
acaagttttt tctatcgtta tgggtttgtt gccaatttca gcatggagct



tcccagcttt





1561
ggggtgtctg ggatcaacga gtctgcggac atgagtattg gagttactgt



catcaaaaac





1621
aatatgataa acaatgatct tggtccagca accgctcaaa tggcccttca



gctgttcatc





1681
aaagattaca ggtacacgta ccggtgccat agaggtgaca cacaaataca



aacccgaaga





1741
tcatttgaaa taaagaaact gtgggagcaa acccattcca aagctggact



gctggtctcc





1801
gacggaggcc caaatttata caacattaga aatctccaca ttcctgaagt



ctgcttgaaa





1861
tgggaattaa tggatgagga ttaccagggg cgtttatgca acccactgaa



cccatttgtc





1921
aaccataaag acattgaatc agtgaacaat gcagtgataa tgccagcaca



tggtccagcc





1981
aaaaacatgg agtatgatgc tgttgcaaca acacactcct ggatccccaa



aagaaatcga





2041
tccatcttga atacaagcca aagaggaata cttgaagatg aacaaatgta



ccaaaagtgc





2101
tgcaacttat ttgaaaaatt cttccccagc agttcataca gaagaccagt



cgggatatcc





2161
agtatggtgg aggctatggt ttccagagcc cgaattgatg cacgaattga



tttcgaatct





2221
ggaaggataa agaaagagga gttcactgag atcatgaaga tctgttccac



cattgaagag





2281
ctcagacggc aaaaatagtg aatttagctt gtccttcatg aaaaaatgcc



ttgtttctac





2341
t











>PA (SEQ ID NO: 51)










1
agcgaaagca ggtactgatt caaaatggaa gattttgtgc gacaatgctt




caatccgatg





61
attgtcgagc ttgcggaaaa ggcaatgaaa gagtatggag aggacctgaa



aatcgaaaca





121
aacaaatttg cagcaatatg cactcacttg gaagtgtgct tcatgtattc



agattttcac





181
ttcatcgatg agcaaggcga gtcaatagtc gtagaacttg gcgatccaaa



tgcacttttg





241
aagcacagat ttgaaataat cgagggaaga gatcgcacaa tagcctggac



agtaataaac





301
agtatttgca acactacagg ggctgagaaa ccaaagtttc taccagattt



gtatgattac





361
aagaagaata gattcatcga aattggagta acaaggagag aagttcacat



atactatctg





421
gaaaaggcca ataaaattaa atctgagaag acacacatcc acattttctc



attcactggg





481
gaggaaatgg ccacaaaggc cgactacact ctcgatgaag aaagcagggc



taggatcaaa





541
accaggctat tcaccataag acaagaaatg gctagcagag gcctctggga



ttcctttcgt





601
cagtccgaga gaggcgaaga gacaattgaa gaaagatttg aaatcacagg



aacaatgcgc





661
aagcttgccg accaaagtct cccgccaaac ttctccagcc ttgaaaaatt



tagagcctat





721
gtggatggat tcgaaccgaa cggctacatt gagggcaagc tttctcaaat



gtccaaagaa





781
gtaaatgcta gaattgaacc ttttttgaaa tcaacaccac gaccacttag



acttccggat





841
gggcctccct gttctcagcg gtccaaattc ctgctgatgg atgccttaaa



attaagcatt





901
gaggacccaa gtcatgaggg agaggggata ccgctatatg atgcaatcaa



atgcatgaga





961
acattctttg gatggaagga acccaatgtt gttaaaccac acgaaaaggg



aataaatcca





1021
aattatcttc tgtcatggaa gcaagtactg gcagaactgc aggacattga



gaatgaggag





1081
aaaattccaa ggactaaaaa tatgaagaaa acgagtcagt taaagtgggc



acttggtgag





1141
aacatggcac cagaaaaggt agactttgac gattgtaaag atgtaggcga



tttgaagcaa





1201
tatgatagtg atgaaccaga attgaggtcg cttgcaagtt ggattcagaa



tgagttcaac





1261
aaggcatgtg aactgaccga ttcaagctgg atagagctcg atgagattgg



agaagatgcg





1321
gctccaattg aacacattgc aagcatgaga aggaattatt tcacagcaga



ggtgtctcat





1381
tgcagagcca cagaatacat aatgaagggg gtgtacatca atactgcctt



gcttaatgca





1441
tcctgtgcag caatggatga tttccaatta attccaatga taagcaagtg



tagaactaag





1501
gagggaaggc gaaagaccaa tttgtacggt ttcatcataa aaggaagatc



ccacttaagg





1561
aatgacaccg atgtggtaaa ctttgtgagc atggagtttt ccctcactga



cccaagactt





1621
gaaccacaca aatgggagaa gtactgtgtt cttgaggtag gagatatgct



tctaagaagt





1681
gccataggcc atgtgtcaag gcctatgttc ttgtatgtga ggacaaatgg



aacctcaaaa





1741
attaaaatga aatgggggat ggaaatgagg cgttgcctcc ttcagtcact



tcaacaaatc





1801
gagagtatga ttgaagctga gtcctctgtc aaggagaaag acatgaccaa



agagttcttt





1861
gaaaacaaat cagaaacatg gcccgttgga gagtccccca aaggagtgga



ggaaggttcc





1921
attgggaagg tctgcagaac tttattggca aagtcggtat tcaacagctt



gtatgcatct





1981
ccacaactag aaggattttc agctgaatca agaaaactgc ttcttatcgt



tcaggctctt





2041
agggacaacc tggaacctgg gacctttgat cttggggggc tatatgaagc



aattgaggag





2101
tgcctgatta atgatccctg ggttttgctt aatgcttctt ggttcaactc



cttcctcaca





2161
catgcattga gatagttgtg gcaatgctac tatttgctat ccatactgtc



caaaaaagta





2221
ccttgtttct act











>NP (SEQ ID NO: 52)










1
agcaaaagca gggtagataa tcactcacag agtgacatcg aaatcatggc




gaccaaaggc





61
accaaacgat cttacgaaca gatggagact gatggagaac gccagaatgc



cactgaaatc





121
agagcatctg tcggaaaaat gattgatgga attggacgat tctacatcca



aatgtgcacc





181
gaacttaaac tcagtgatta tgagggacgg ctgattcaga acagcttaac



aatagagaga





241
atggtgctct ctgcttttga cgagaggagg aataaatatc tagaagaaca



tcccagtgcg





301
gggaaagatc ctaagaaaac tggaggacct atatacagga gagtagatgg



aaagtggagg





361
agagaactca tcctttatga caaagaagaa ataagacgaa tctggcgcca



agctaataat





421
ggtgacgatg caacggctgg tctgactcac atgatgatct ggcactccaa



tttgaatgat





481
gcaacttacc agaggacaag agctcttgtt cgcacaggaa tggatcccag



gatgtgctca





541
ctgatgcagg gttcaaccct ccctaggagg tctggggccg caggtgctgc



agtcaaagga





601
gttggaacaa tggtgatgga attgatcaga atgatcaaac gtgggatcaa



tgatcggaac





661
ttctggaggg gtgagaatgg acggagaaca aggattgctt atgaaagaat



gtgcaacatt





721
ctcaaaggga aatttcaaac agctgcacaa agaacaatgg tggatcaagt



gagagagagc





781
cggaatccag gaaatgctga gttcgaagat ctcatctttt tagcacggtc



tgcactcata





841
ttgagagggt cagttgctca caagtcctgc ctgcctgcct gtgtgtatgg



atctgccgta





901
gccagtggat acgactttga aagagaggga tactctctag tcggaataga



ccctttcaga





961
ctgcttcaaa acagccaagt atacagccta atcagaccaa atgagaatcc



agcacacaag





1021
agtcaactgg tgtggatggc atgccattct gctgcatttg aagatctaag



agtatcaagc





1081
ttcatcagag ggacgaaagt ggtcccaaga gggaagcttt ccactagagg



agttcaaatt





1141
gcttccaatg aaaacatgga gactatggaa tcaagtaccc ttgaactgag



aagcagatac





1201
tgggccataa ggaccagaag tggagggaac accaatcaac agagggcttc



ctcgggccaa





1261
atcagcatac aacctacgtt ctcagtacag agaaatctcc cttttgacag



accaaccatt





1321
atggcagcat tcactgggaa tacagagggg agaacatctg acatgagaac



cgaaatcata





1381
aggctgatgg aaagtgcaag accagaagat gtgtctttcc aggggcgggg



agtcttcgag





1441
ctctcggacg aaaaggcaac gagcccgatc gtgccctcct ttgacatgag



taatgaagga





1501
tcttatttct tcggagacaa tgcagaggag tacgacaatt aaagaaaaat



acccttgttt





1561
ctact











>HA (SEQ ID NO: 53)










1
agcaaaagca ggggaaaata aaaacaacca aaatgaaggc aaaactactg




gtcctgttat





61
atgcatttgt agctacagat gcagacacaa tatgtatagg ctaccatgcg



aacaactcaa





121
ccgacactgt tgacacaata ctcgagaaga atgtggcagt gacacattct



gttaacctgc





181
tcgaagacag ccacaacggg aaactatgta aattaaaagg aatagcccca



ctacaattgg





241
ggaaatgtaa catcaccgga tggctcttgg gaaatccaga atgcgactca



ctgcttccag





301
cgagatcatg gtcctacatt gtagaaacac caaactctga gaatggagca



tgttatccag





361
gagatctcat cgactatgag gaactgaggg agcaattgag ctcagtatca



tcattagaaa





421
gattcgaaat atttcccaag gaaagttcat ggcccaacca cacattcaac



ggagtaacag





481
tatcatgctc ccatagggga aaaagcagtt tttacagaaa tttgctatgg



ctgacgaaga





541
agggggattc atacccaaag ctgaccaatt cctatgtgaa caataaaggg



aaagaagtcc





601
ttgtactatg gggtgttcat cacccgtcta gcagtgatga gcaacagagt



ctctatagta





661
atggaaatgc ttatgtctct gtagcgtctt caaattataa caggagattc



accccggaaa





721
tagctgcaag gcccaaagta agagatcaac atgggaggat gaactattac



tggaccttgc





781
tagaacccgg agacacaata atatttgagg caactggtaa tctaatagca



ccatggtatg





841
ctttcgcact gagtagaggg tttgagtccg gcatcatcac ctcaaacgcg



tcaatgcatg





901
agtgtaacac gaagtgtcaa acaccccagg gagctataaa cagcaatctc



cctttccaga





961
atatacaccc agtcacaata ggagagtgcc caaaatatgt caggagtacc



aaattgagga





1021
tggttacagg actaagaaac atcccatcca ttcaatacag aggtctattt



ggagccattg





1081
ctggttttat tgagggggga tggactggaa tgatagatgg atggtatggt



tatcatcatc





1141
agaatgaaca gggatcaggc tatgcagcgg atcaaaaaag cacacaaaat



gccattaacg





1201
ggattacaaa caaggtgaac tctgttatcg agaaaatgaa cactcaattc



acagctgtgg





1261
gtaaagaatt caacaactta gaaaaaagga tggaaaattt aaataaaaaa



gttgatgatg





1321
ggtttctgga catttggaca tataatgcag aattgttagt tctactggaa



aatgaaagga





1381
ctttggattt ccatgactta aatgtgaaga atctgtacga gaaagtaaaa



agccaattaa





1441
agaataatgc caaagaaatc ggaaatgggt gttttgagtt ctaccacaag



tgtgacaatg





1501
aatgcatgga aagtgtaaga aatgggactt atgattatcc aaaatattca



gaagaatcaa





1561
agttgaacag ggaaaagata gatggagtga aattggaatc aatgggggtg



tatcagattc





1621
tggcgatcta ctcaactgtc gccagttcac tggtgctttt ggtctccctg



ggggcaatca





1681
gtttctggat gtgttctaat gggtctttgc agtgcagaat atgcatctga



gattaggatt





1741
tcagaaatat aaggaaaaac acccttgttt ctact











>NA (SEQ ID: 54)










1
agcgaaagca ggagtttaaa tgaatccaaa ccagaaaata ataaccattg




ggtcaatctg





61
tatggtagtc ggaataatta gcctaatatt gcaaatagga aatataatct



caatatggat





121
tagccattca attcaaaccg gaaatcaaaa ccatactgga atatgcaacc



aaggcagcat





181
tacctataaa gttgttgctg ggcaggactc aacttcagtg atattaaccg



gcaattcatc





241
tctttgtccc atccgtgggt gggctataca cagcaaagac aatggcataa



gaattggttc





301
caaaggagac gtttttgtca taagagagcc ttttatttca tgttctcact



tggaatgcag





361
gacctttttt ctgactcaag gcgccttact gaatgacaag cattcaaggg



ggacctttaa





421
ggacagaagc ccttataggg ccttaatgag ctgccctgtc ggtgaagctc



cgtccccgta





481
caattcaagg tttgaatcgg ttgcttggtc agcaagtgca tgtcatgatg



gaatgggctg





541
gctaacaatc ggaatttctg gtccagatga tggagcagtg gctgtattaa



aatacaaccg





601
cataataact gaaaccataa aaagttggag gaagaatata ttgagaacac



aagagtctga





661
atgtacctgt gtaaatggtt catgttttac cataatgacc gatggcccaa



gtgatgggct





721
ggcctcgtac aaaattttca agatcgagaa ggggaaggtt actaaatcga



tagagttgaa





781
tgcacctaat tctcactacg aggaatgttc ctgttaccct gataccggca



aagtgatgtg





841
tgtgtgcaga gacaattggc acggttcgaa ccgaccatgg gtgtccttcg



accaaaacct





901
agattataaa ataggataca tctgcagtgg ggttttcggt gacaacccgc



gtcccaaaga





961
tggaacaggc agctgtggcc cagtgtctgc tgatggagca aacggagtaa



agggattttc





1021
atataagtat ggcaatggtg tttggatagg aaggactaaa agtgacagtt



ccagacatgg





1081
gtttgagatg atttgggatc ctaatggatg gacagagact gatagtaggt



tctctatgag





1141
acaagatgtt gtggcaataa ctaatcggtc agggtacagc ggaagtttcg



ttcaacatcc





1201
tgagctaaca gggctagact gtatgaggcc ttgcttctgg gttgaattaa



tcagggggct





1261
acctgaggag gacgcaatct ggactagtgg gagcatcatt tctttttgtg



gtgtgaatag





1321
tgatactgta gattggtctt ggccagacgg tgctgagttg ccgttcacca



ttgacaagta





1381
gtttgttcaa aaaactcctt gtttctact











>M (SEQ ID NO: 55)










1
agcaaaagca ggtagatatt gaaagatgag tcttctaacc gaggtcgaaa




cgtacgttct





61
ctctatcgtc ccgtcaggcc ccctcaaagc cgagatcgca cagagacttg



aagatgtctt





121
tgcagggaag aacaccgatc ttgaggttct catggaatgg ctaaagacaa



gaccaatcct





181
gtcacctctg actaagggga ttttaggatt tgtgttcacg ctcaccgtgc



ccagtgagcg





241
gggactgcag cgtagacgct ttgtccaaaa tgctcttaat gggaacggag



atccaaataa





301
catggacaaa gcagttaaac tgtataggaa gcttaagagg gagataacat



tccatggggc





361
caaagaaata gcactcagtt attctgctgg tgcacttgcc agttgtatgg



gcctcatata





421
caacaggatg ggggctgtga ccactgaagt ggcatttggc ctggtatgcg



caacctgtga





481
acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa



ccaatccact





541
aatcagacat gagaacagaa tggttctagc cagcactaca gctaaggcta



tggagcaaat





601
ggctggatcg agtgagcaag cagcagaggc catggatatt gctagtcagg



ccaggcaaat





661
ggtgcaggcg atgagaaccg ttgggactca tcctagctcc agtgctggtc



taaaagatga





721
tcttcttgaa aatttgcagg cctatcagaa acgaatgggg gtgcagatgc



aacgattcaa





781
gtgatcctct cgtcattgca gcaaatatca ttggaatctt gcacttgata



ttgtggattc





841
ttgatcgtct ttttttcaaa tgcatttatc gtcgctttaa atacggtttg



aaaagagggc





901
cttctacgga aggagtgcca gagtctatga gggaagaata tcgaaaggaa



cagcagaatg





961
ctgtggatgt tgacgatggt cattttgtca acatagagct ggagtaaaaa



actaccttgt





1021
ttctact











>NS (SEQ ID NO: 56)










1
agcaaaagca gggtgacaaa gacataatgg atccaaacac tgtgtcaagc




tttcaggtag





61
attgctttct ttggcatgtc cgcaaaagag ttgcagacca agaactaggt



gatgccccat





121
tccttgatcg gcttcgccga gatcagaagt ccctaagagg aagaggcagc



actcttggtc





181
tggacatcga aacagccacc cgtgctggaa agcaaatagt ggagcggatt



ctgaaggaag





241
aatctgatga ggcactcaaa atgaccatgg cctctgtacc tgcatcgcgc



tacctaactg





301
acatgactct tgaggaaatg tcaaggcact ggttcatgct catgcccaag



cagaaagtgg





361
caggccctct ttgtatcaga atggaccagg cgatcatgga taagaacatc



atactgaaag





421
cgaacttcag tgtgattttt gaccggctgg agactctaat attactaagg



gccttcaccg





481
aagaggggac aattgttggc gaaatttcac cactgccctc tcttccagga



catactgatg





541
aggatgtcaa aaatgcagtt ggggtcctca tcggaggact tgaatggaat



aataacacag





601
ttcgagtctc tgaaactcta cagagattcg cttggagaag cagtaatgag



aatgggagac





661
ctccactcac tccaaaacag aaacggaaaa tggcgggaac aattaggtca



gaagtttgaa





721
gaaataagat ggttgattga agaagtgaga cacagactga agataacaga



gaatagtttt





781
gagcaaataa catttatgca agccttacaa ctattgcttg aagtggagca



agagataaga





841
actttctcgt ttcagcttat ttaataataa aaaacaccct tgtttctact






Plasmid Sequences










1. Plasmid pYA4379 (SEQ ID NO: 57)



Ampicillin resistance gene (amp): complement(4837 . . . 5697)


BGH polyA signal 1433 . . . 1657


CMV promoter 232 . . . 819


Neomycin resistance gene (neo): 2541 . . . 3335


pUC ori complement(4022 . . . 4692)


Chicken PolI promoter (CPI): complement(968 . . . 1382)


Murine PolI terminator (MTI): 901 . . . 941












1
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg






61
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg





121
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc





181
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt





241
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata





301
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc





361
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc





421
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt





481
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt





541
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca





601
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg





661
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc





721
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg





781
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca





841
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc





901
gtgtcgcccg gagtactggt cgacctccga agttgggggg gagcagcagg tggtaccacc





961
tgctcctaca gacgaacata taaggcatcc gaaaaaaacg ttctagtccc ataggcgccg





1021
actaccggca gcggctccga cggcagccga ggtttacctc gacgtaactg gaggtacaaa





1081
attacagcga cgcctctggc agctccggag ctgtagcgcc cccccccaca gccagagcgg





1141
ccaagacaat ccgaaacggg gtagacctgg acgcggatcg caagccgccc cggcagcgac





1201
ctctagccgc cgccgcggag agcgcgagac ggtagcaccc gggtagaccg ttccgccgtt





1261
tccgagacgc cccggcagcg acccctagcc gccgccgccg cggagagacc gagccggacg





1321
gtgcccgccg ggaccaggta gaccgttccg ccgtgcccca gccacctccg cgaagcgacc





1381
gaaagggcga attctgcaga aagcttaagt ttaaaccgct gatcagcctc gactgtgcct





1441
tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt





1501
gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg





1561
tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac





1621
aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc





1681
tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg





1741
gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct





1801
ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg





1861
ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag





1921
ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg





1981
gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc





2041
tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat





2101
gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt





2161
gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt





2221
cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc





2281
atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc





2341
cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg





2401
ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc





2461
taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga





2521
caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg





2581
cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg





2641
ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt





2701
ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg





2761
gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat





2821
tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat





2881
ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg





2941
accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg





3001
atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc





3061
tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc





3121
cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg





3181
tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg





3241
gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca





3301
tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac





3361
cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga





3421
aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga





3481
tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa





3541
ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg





3601
tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta





3661
gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat





3721
tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag





3781
ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg





3841
ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc





3901
ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc





3961
agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa





4021
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt





4081
tttccatagg ctccgcccccc tgacgagca tcacaaaaat cgacgctcaa gtcagaggtg





4141
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg





4201
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag





4261
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc





4321
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa





4381
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg





4441
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc





4501
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac





4561
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggttt





4621
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat





4681
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat





4741
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc





4801
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc





4861
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta





4921
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga





4981
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg





5041
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc





5101
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat





5161
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag





5221
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat





5281
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa





5341
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa





5401
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga





5461
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg





5521
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc





5581
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg





5641
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact





5701
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat





5761
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt





5821
gccacctgac gtc











2. Plasmid pYA4380 (SEQ ID NO: 58)



Ampicillin resistance gene (amp): complement(4191 . . . 5051)


BGH gene polyA signal 787 . . . 1011


Neomycin resistance gene (neo): 1895 . . . 2689


pUC ori complement(3376 . . . 4046)


Murine PolI terminator (MTI): 255 . . . 295


chicken RNA PolI promoter(CPI): complement(322 . . . 736)












1
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg






61
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg





121
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc





181
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt





241
gattattgac tagcgtgtcg cccggagtac tggtcgacct ccgaagttgg gggggagcag





301
caggtggtac cacctgctcc tacagacgaa catataaggc atccgaaaaa aacgttctag





361
tcccataggc gccgactacc ggcagcggct ccgacggcag ccgaggttta cctcgacgta





421
actggaggta caaaattaca gcgacgcctc tggcagctcc ggagctgtag cgcccccccc





481
cacagccaga gcggccaaga caatccgaaa cggggtagac ctggacgcgg atcgcaagcc





541
gccccggcag cgacctctag ccgccgccgc ggagagcgcg agacggtagc acccgggtag





601
accgttccgc cgtttccgag acgccccggc agcgacccct agccgccgcc gccgcggaga





661
gaccgagccg gacggtgccc gccgggacca ggtagaccgt tccgccgtgc cccagccacc





721
tccgcgaagc gaccgaaagg gcgaattctg cagaaagctt aagtttaaac cgctgatcag





781
cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct





841
tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc





901
attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg





961
aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg





1021
cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa





1081
gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc





1141
ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag





1201
ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca





1261
aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc





1321
gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa





1381
cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct





1441
attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt





1501
gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat





1561
gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag





1621
tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat





1681
cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt





1741
tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg





1801
cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg





1861
atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc





1921
aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat





1981
cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt





2041
caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg





2101
gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag





2161
ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc





2221
tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc





2281
tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga





2341
agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga





2401
actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg





2461
cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg





2521
tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc





2581
tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc





2641
cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg





2701
gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc





2761
gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc





2821
ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct





2881
tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca





2941
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg





3001
tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt





3061
tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt





3121
gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg





3181
ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg





3241
cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg





3301
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat





3361
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc





3421
gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc





3481
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga





3541
agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt





3601
ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg





3661
taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc





3721
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg





3781
gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc





3841
ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg





3901
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc





3961
gctggtagcg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa





4021
gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa





4081
gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa





4141
tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc





4201
ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga





4261
ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca





4321
atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc





4381
ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat





4441
tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc





4501
attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt





4561
tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc





4621
ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg





4681
gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt





4741
gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg





4801
gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga





4861
aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg





4921
taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg





4981
tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt





5041
tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc





5101
atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca





5161
tttccccgaa aagtgccacc tgacgtc











3. Plasmid pYA4749 (SEQ ID NO: 59)



Chloramphenicol resistance gene (cat): complement (3519 . . . 219)


p15A ori: 581 . . . 1429


GFP Gene: 1800 . . . 2516


Ptrc promoter: 1638 . . . 1740


5ST1T2 terminator: 2549 . . . 3052












1
gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt






61
gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt





121
ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga





181
tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga





241
aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt





301
ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc





361
ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat





421
ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt





481
gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg





541
acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgctagcgg agtgtatact





601
ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa





661
aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc





721
actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc





781
ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa





841
agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc





901
agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc





961
tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc





1021
gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac





1081
tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt





1141
gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt





1201
agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg





1261
tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt





1321
cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc





1381
aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tcgtccattc





1441
cgacagcatc gccagtcact atggcgtgct gctagcgcta tatgcgttga tgcaatttct





1501
atgcgcaccc gttctcggag cactgtccga ccgctttggc cgccgcccag tcctgctcgc





1561
ttcgctactt ggagccacta tcgactacgc gatcatggcg accacacccg tcctgtgtaa





1621
tacgtagaca ctgtgtctcc ggaagacctt ccattctgaa atgagctgtt gacaattaat





1681
catccggctc gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacaga





1741
ccatgggaat tcgagctcgg tacccgggga tcctctagat ttaagaagga gatatacata





1801
tgagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg





1861
atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa





1921
aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg





1981
tcactacttt cgcgtatggt cttcaatgct ttgcgagata cccagatcat atgaaacagc





2041
atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca





2101
aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta





2161
atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat





2221
tggaatacaa ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa





2281
tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc





2341
attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc





2401
tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc





2461
ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac aaataaatgt





2521
ccagacctgc agccaagctc ccaagcttgg ctgttttggc ggatgagaga agattttcag





2581
cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg





2641
cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc





2701
cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac





2761
gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc





2821
tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag





2881
ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc





2941
tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta aatacattca





3001
aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataatg gagacacagt





3061
gtcagatctt aaccggcagc gcccaacagt cccccggcca cggggcctgc caccataccc





3121
acgccgaaac aagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg





3181
ctaccctgtg gaacacctac atctgtatta acgaagcgct aaccgttttt atcaggctct





3241
gggaggcaga ataaatgatc atatcgtcaa ttattacctc cacggggaga gcctgagcaa





3301
actggcctca ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg





3361
taaaccagca atagacataa gcggctattt aacgaccctg ccctgaaccg acgaccgggt





3421
cgaatttgct ttcgaatttc tgccattcat ccgcttatta tcacttattc aggcgtagca





3481
ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca





3541
tcgcagtact gttgtaattc attaagcatt ctgccgacat ggaagccatc acagacggca





3601
tgatgaacct gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc





3661
atggtgaaaa cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg





3721
aaactcaccc agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa





3781
taggccaggt tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg





3841
aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg





3901
gtgtaacaag ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacg











IV. The 8-unit plasmid pYA4519 (SEQ ID NO: 60)



CPI: complement (5606 . . . 6020)


CPI: complement (7234 . . . 7648)


CPI: complement (10706 . . . 11120)


CPI: complement (12472 . . . 12886)


CPI: complement (15836 . . . 16250)


CPI: complement (17984 . . . 18398)


CPI: complement (20680 . . . 21094)


CPI: complement (23204 . . . 23618)


MTI: 3224 . . . 3264


MTI: 6303 . . . 6343


MTI: 8324 . . . 8364


MTI: 13562 . . . 13602


MTI: 16534 . . . 16574


MTI: 19074 . . . 19114


MTI: 11404 . . . 11444


MTI: 21388 . . . 21428


CMV: 7655 . . . 8242


CMV: 2556 . . . 3142


CMV: 12893 . . . 13480


CMV: 18405 . . . 18992


BGH: 6071 . . . 6295


BGH: 11171 . . . 11395


BGH: 16301 . . . 16525


BGH: 21145 . . . 21369


PB2: 3265 . . . 5605


PB1: 8365 . . . 10705


PA: 13603 . . . 15835


NP: 19115 . . . 20679


HA: 21429 . . . 23203


NA: 16575 . . . 17983


M: 11445 . . . 12471


NS: 6344 . . . 7233


Chloramphenicol resistance gene (cat): 1423 . . . 2082


p15A ori: complement (213 . . . 1061)












1
gccggctaaa gtgtctacgt attacacagg acgggtgtgg tcgccatgat cgcgtagtcg






61
atagtggctc caagtagcga agcgagcagg actgggcggc ggccaaagcg gtcggacagt





121
gctccgagaa cgggtgcgca tagaaattgc atcaacgcat atagcgctag cagcacgcca





181
tagtgactgg cgatgctgtc ggaatggacg atctagaaat attttatctg attaataaga





241
tgatcttctt gagatcgttt tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc





301
gccttgcagg gcggtttttc gaaggttctc tgagctacca actctttgaa ccgaggtaac





361
tggcttggag gagcgcagtc accaaaactt gtcctttcag tttagcctta accggcgcat





421
gacttcaaga ctaactcctc taaatcaatt accagtggct gctgccagtg gtgcttttgc





481
atgtctttcc gggttggact caagacgata gttaccggat aaggcgcagc ggtcggactg





541
aacggggggt tcgtgcatac agtccagctt ggagcgaact gcctacccgg aactgagtgt





601
caggcgtgga atgagacaaa cgcggccata acagcggaat gacaccggta aaccgaaagg





661
caggaacagg agagcgcacg agggagccgc cagggggaaa cgcctggtat ctttatagtc





721
ctgtcgggtt tcgccaccac tgatttgagc gtcagatttc gtgatgcttg tcaggggggc





781
ggagcctatg gaaaaacggc tttgccgcgg ccctctcact tccctgttaa gtatcttcct





841
ggcatcttcc aggaaatctc cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac





901
gaccgagcgt agcgagtcag tgagcgagga agcggaatat atcctgtatc acatattctg





961
ctgacgcacc ggtgcagcct tttttctcct gccacatgaa gcacttcact gacaccctca





1021
tcagtgccaa catagtaagc cagtatacac tccgctagcg ctgatgtccg gcggtgcttt





1081
tgccgttacg caccaccccg tcagtagctg aacaggaggg acagctgata gaaacagaag





1141
ccactggagc acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc





1201
gacgcacttt gcgccgaata aatacctgtg acggaagatc acttcgcaga ataaataaat





1261
cctggtgtcc ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt





1321
gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact accgggcgta





1381
ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact





1441
ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag





1501
tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag





1561
accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg





1621
atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt gatatgggat





1681
agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg





1741
agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt





1801
tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca





1861
gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc





1921
ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg





1981
ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaat





2041
gaattacaac agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat





2101
tggtgccctt aaacgcctgg tgctacgcct gaataagtga taataagcgg atgaatggca





2161
gaaattcgaa agcaaattcg acccggtcgt cggttcaggg cagggtcgtt aaatagccgc





2221
ttatgtctat tgctggttta ccggtttatt gactaccgga agcagtgtga ccgtgtgctt





2281
ctcaaatgcc tgaggccagt ttgctcaggc tctccccgtg gaggtaataa ttgacgatat





2341
gatcatttat tctgcctccc agagcctgat aaaaacggtt agcgcttcgt taatacagat





2401
gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg





2461
cagggcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc





2521
gctgccggtt aagatctgac acttaagccc gggcgttgac attgattatt gactagttat





2581
taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca





2641
taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca





2701
ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg





2761
gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg





2821
ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc





2881
ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg





2941
atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca





3001
agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt





3061
ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg





3121
gaggtctata taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc





3181
gaaattaata cgactcacta tagggagacc caagctggct agcgtgtcgc ccggagtact





3241
ggtcgacctc cgaagttggg ggggagcgaa agcaggtcaa ttatattcaa tatggaaaga





3301
ataaaagaac taaggaatct aatgtcgcag tctcgcactc gcgagatact cacaaaaacc





3361
accgtggacc atatggccat aatcaagaag tacacatcag gaagacagga gaagaaccca





3421
gcacttagga tgaaatggat gatggcaatg aaatatccaa ttacagcaga caagaggata





3481
acggaaatga ttcctgagag aaatgagcag ggacaaactt tatggagtaa aatgaatgac





3541
gccggatcag accgagtgat ggtatcacct ctggctgtga catggtggaa taggaatgga





3601
ccagtgacaa gtacagttca ttatccaaaa atctacaaaa cttattttga aaaagtcgaa





3661
aggttaaaac atggaacctt tggccctgtc cattttagaa accaagtcaa aatacgtcga





3721
agagttgaca taaatcctgg tcatgcagat ctcagtgcca aagaggcaca ggatgtaatc





3781
atggaagttg ttttccctaa cgaagtggga gccaggatac taacatcgga atcgcaacta





3841
acgacaacca aagagaagaa agaagaactc cagggttgca aaatttctcc tctgatggtg





3901
gcatacatgt tggagagaga actggtccgc aaaacgagat tcctcccagt ggctggtgga





3961
acaagcagtg tgtacattga agtgttgcat ttgacccaag gaacatgctg ggaacagatg





4021
tacactccag gaggggaggc gaggaatgat gatgttgatc aaagcttaat tattgctgct





4081
agaaacatag taagaagagc cacagtatca gcagatccac tagcatcttt attggagatg





4141
tgccacagca cgcagattgg tggaataagg atggtaaaca tccttaggca gaacccaaca





4201
gaagagcaag ccgtggatat ttgcaaggct gcaatgggac tgagaattag ctcatccttc





4261
agttttggtg gattcacatt taagagaaca agcggatcat cagtcaagag agaggaagag





4321
gtgcttacgg gcaatcttca gacattgaag ataagagtac atgagggata tgaagagttc





4381
acaatggttg ggagaagagc aacagctata ctcagaaaag caaccaggag attgattcag





4441
ctgatagtga gtgggagaga cgaacagtcg attgccgaag caataattgt ggccatggta





4501
ttttcacaag aggattgtat gataaaagca gttagaggtg acctgaattt cgtcaatagg





4561
gcgaatcagc gattgaatcc catgcaccaa cttttgagac attttcagaa ggatgcaaag





4621
gtgctctttc aaaattgggg aattgaatcc atcgacaatg tgatgggaat gatcgggata





4681
ttgcccgaca tgactccaag caccgagatg tcaatgagag gagtgagaat cagcaaaatg





4741
ggggtagatg agtattccag cgcggagaag atagtggtga gcattgaccg ttttttgaga





4801
gttagggacc aacgtgggaa tgtactactg tctcccgagg agatcagtga aacacaggga





4861
acagagaaac tgacaataac ttactcatcg tcaatgatgt gggagattaa tggtcctgaa





4921
tcagtgttgg tcaataccta tcagtggatc atcagaaact gggaaactgt taaaattcag





4981
tggtcccaga atcctacaat gctgtacaat aaaatggaat ttgagccatt tcagtcttta





5041
gttccaaagg ccgttagagg ccaatacagt gggtttgtga gaactctgtt ccaacaaatg





5101
agggatgtgc ttgggacatt tgataccgct cagataataa aacttcttcc cttcgcagcc





5161
gctccaccaa agcaaagtag aacgcagttc tcctcattga ctataaatgt gaggggatca





5221
ggaatgagaa tacttgtaag gggcaattct ccagtattca actacaacaa gaccactaaa





5281
agactcacag ttctcggaaa ggatgctggc cctttaactg aagacccaga tgaaggcaca





5341
gctggagttg agtccgcagt tctgagagga ttcctcattc tgggcaaaga agacaggaga





5401
tatggaccag cattaagcat aaatgaactg agcaaccttg cgaaaggaga gaaggctaat





5461
gtgctaattg ggcaaggaga cgtggtgttg gtaatgaaac ggaaacggaa ctctagcata





5521
cttactgaca gccagacagc gaccaaaaga attcggatgg ccatcaatta gtgtcgaata





5581
gtttaaaaac gaccttgttt ctactacaga cgaacatata aggcatccga aaaaaacgtt





5641
ctagtcccat aggcgccgac taccggcagc ggctccgacg gcagccgagg tttacctcga





5701
cgtaactgga ggtacaaaat tacagcgacg cctctggcag ctccggagct gtagcgcccc





5761
cccccacagc cagagcggcc aagacaatcc gaaacggggt agacctggac gcggatcgca





5821
agccgccccg gcagcgacct ctagccgccg ccgcggagag cgcgagacgg tagcacccgg





5881
gtagaccgtt ccgccgtttc cgagacgccc cggcagcgac ccctagccgc cgccgccgcg





5941
gagagaccga gccggacggt gcccgccggg accaggtaga ccgttccgcc gtgccccagc





6001
cacctccgcg aagcgaccga aagggcgaat tctgcagaaa gcttaagttt aaaccgctga





6061
tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct





6121
tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca





6181
tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag





6241
ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcggcc





6301
gcgtgtcgcc cggagtactg gtcgacctcc gaagttgggg gggagcaaaa gcagggtgac





6361
aaagacataa tggatccaaa cactgtgtca agctttcagg tagattgctt tctttggcat





6421
gtccgcaaaa gagttgcaga ccaagaacta ggtgatgccc cattccttga tcggcttcgc





6481
cgagatcaga agtccctaag aggaagaggc agcactcttg gtctggacat cgaaacagcc





6541
acccgtgctg gaaagcaaat agtggagcgg attctgaagg aagaatctga tgaggcactc





6601
aaaatgacca tggcctctgt acctgcatcg cgctacctaa ctgacatgac tcttgaggaa





6661
atgtcaaggc actggttcat gctcatgccc aagcagaaag tggcaggccc tctttgtatc





6721
agaatggacc aggcgatcat ggataagaac atcatactga aagcgaactt cagtgtgatt





6781
tttgaccggc tggagactct aatattacta agggccttca ccgaagaggg gacaattgtt





6841
ggcgaaattt caccactgcc ctctcttcca ggacatactg atgaggatgt caaaaatgca





6901
gttggggtcc tcatcggagg acttgaatgg aataataaca cagttcgagt ctctgaaact





6961
ctacagagat tcgcttggag aagcagtaat gagaatggga gacctccact cactccaaaa





7021
cagaaacgga aaatggcggg aacaattagg tcagaagttt gaagaaataa gatggttgat





7081
tgaagaagtg agacacagac tgaagataac agagaatagt tttgagcaaa taacatttat





7141
gcaagcctta caactattgc ttgaagtgga gcaagagata agaactttct cgtttcagct





7201
tatttaataa taaaaaacac ccttgtttct actacagacg aacatataag gcatccgaaa





7261
aaaacgttct agtcccatag gcgccgacta ccggcagcgg ctccgacggc agccgaggtt





7321
tacctcgacg taactggagg tacaaaatta cagcgacgcc tctggcagct ccggagctgt





7381
agcgcccccc cccacagcca gagcggccaa gacaatccga aacggggtag acctggacgc





7441
ggatcgcaag ccgccccggc agcgacctct agccgccgcc gcggagagcg cgagacggta





7501
gcacccgggt agaccgttcc gccgtttccg agacgccccg gcagcgaccc ctagccgccg





7561
ccgccgcgga gagaccgagc cggacggtgc ccgccgggac caggtagacc gttccgccgt





7621
gccccagcca cctccgcgaa gcgaccgagc gcgcgttgac attgattatt gactagttat





7681
taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca





7741
taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca





7801
ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg





7861
gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg





7921
ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc





7981
ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg





8041
atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca





8101
agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt





8161
ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg





8221
gaggtctata taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc





8281
gaaattaata cgactcacta tagggagacc caagctggct agcgtgtcgc ccggagtact





8341
ggtcgacctc cgaagttggg ggggagcgaa agcaggcaaa ccatttgaat ggatgtcaat





8401
ccgactttac ttttcttaaa agtgccagca caaaatgcta taagcacaac tttcccttat





8461
actggagacc ctccttacag ccatgggaca ggaacaggat acaccatgga tactgtcaac





8521
aggacacatc agtactcaga aaggggaaga tggacaacaa acaccgaaac tggagcaccg





8581
caactcaacc cgattgatgg gccactgcca gaagacaatg aaccaagtgg ttatgcccaa





8641
acagattgtg tattggaagc aatggccttc cttgaggaat cccatcctgg tatctttgag





8701
acctcgtgtc ttgaaacgat ggaggttgtt cagcaaacac gagtggacaa gctgacacaa





8761
ggccgacaga cctatgactg gactctaaat aggaaccagc ctgctgcaac agcattggcc





8821
aacacaatag aagtgttcag atcaaatggc ctcacggcca atgaatctgg aaggctcata





8881
gacttcctta aggatgtaat ggagtcaatg aacaaagaag aaatggagat cacaactcat





8941
tttcagagaa agagacgagt gagagacaat atgactaaga aaatggtgac acagagaaca





9001
ataggtaaaa ggaagcagag attgaacaaa aggagttatc taattagggc attaaccctg





9061
aacacaatga ccaaagatgc tgagagaggg aagctaaaac ggagagcaat tgcaacccca





9121
gggatgcaaa taagggggtt tgtatacttt gttgagacac tagcaaggag tatatgtgag





9181
aaacttgaac aatcaggatt gccagttgga ggcaatgaga agaaagcaaa gttggcaaat





9241
gttgtaagga agatgatgac caattctcag gacactgaaa tttctttcac catcactgga





9301
gataacacca aatggaacga aaatcagaac cctcggatgt ttttggccat gatcacatat





9361
ataaccagaa atcagcccga atggttcaga aatgttctaa gtattgctcc aataatgttc





9421
tcaaacaaaa tggcgagact gggaaagggg tacatgtttg agagcaagag tatgaaaatt





9481
agaactcaaa tacctgcaga aatgctagca agcatcgatt tgaaatactt caatgattca





9541
actagaaaga agattgaaaa aatccggccg ctcttaatag atgggactgc atcattgagc





9601
cctggaatga tgatgggcat gttcaatatg ttaagtactg tattaggcgt ctccatcctg





9661
aatcttggac aaaagagaca caccaagact acttactggt gggatggtct tcaatcttct





9721
gatgattttg ctctgattgt gaatgcaccc aatcatgaag ggattcaagc cggagtcaac





9781
aggttttatc gaacctgtaa gctacttgga attaatatga gcaagaaaaa gtcttacata





9841
aacagaacag gtacatttga attcacaagt tttttctatc gttatgggtt tgttgccaat





9901
ttcagcatgg agcttcccag ctttggggtg tctgggatca acgagtctgc ggacatgagt





9961
attggagtta ctgtcatcaa aaacaatatg ataaacaatg atcttggtcc agcaaccgct





10021
caaatggccc ttcagctgtt catcaaagat tacaggtaca cgtaccggtg ccatagaggt





10081
gacacacaaa tacaaacccg aagatcattt gaaataaaga aactgtggga gcaaacccat





10141
tccaaagctg gactgctggt ctccgacgga ggcccaaatt tatacaacat tagaaatctc





10201
cacattcctg aagtctgctt gaaatgggaa ttaatggatg aggattacca ggggcgttta





10261
tgcaacccac tgaacccatt tgtcaaccat aaagacattg aatcagtgaa caatgcagtg





10321
ataatgccag cacatggtcc agccaaaaac atggagtatg atgctgttgc aacaacacac





10381
tcctggatcc ccaaaagaaa tcgatccatc ttgaatacaa gccaaagagg aatacttgaa





10441
gatgaacaaa tgtaccaaaa gtgctgcaac ttatttgaaa aattcttccc cagcagttca





10501
tacagaagac cagtcgggat atccagtatg gtggaggcta tggtttccag agcccgaatt





10561
gatgcacgaa ttgatttcga atctggaagg ataaagaaag aggagttcac tgagatcatg





10621
aagatctgtt ccaccattga agagctcaga cggcaaaaat agtgaattta gcttgtcctt





10681
catgaaaaaa tgccttgttt ctactacaga cgaacatata aggcatccga aaaaaacgtt





10741
ctagtcccat aggcgccgac taccggcagc ggctccgacg gcagccgagg tttacctcga





10801
cgtaactgga ggtacaaaat tacagcgacg cctctggcag ctccggagct gtagcgcccc





10861
cccccacagc cagagcggcc aagacaatcc gaaacggggt agacctggac gcggatcgca





10921
agccgccccg gcagcgacct ctagccgccg ccgcggagag cgcgagacgg tagcacccgg





10981
gtagaccgtt ccgccgtttc cgagacgccc cggcagcgac ccctagccgc cgccgccgcg





11041
gagagaccga gccggacggt gcccgccggg accaggtaga ccgttccgcc gtgccccagc





11101
cacctccgcg aagcgaccga aagggcgaat tctgcagaaa gcttaagttt aaaccgctga





11161
tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct





11221
tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca





11281
tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag





11341
ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcctgc





11401
agggtgtcgc ccggagtact ggtcgacctc cgaagttggg ggggagcaaa agcaggtaga





11461
tattgaaaga tgagtcttct aaccgaggtc gaaacgtacg ttctctctat cgtcccgtca





11521
ggccccctca aagccgagat cgcacagaga cttgaagatg tctttgcagg gaagaacacc





11581
gatcttgagg ttctcatgga atggctaaag acaagaccaa tcctgtcacc tctgactaag





11641
gggattttag gatttgtgtt cacgctcacc gtgcccagtg agcggggact gcagcgtaga





11701
cgctttgtcc aaaatgctct taatgggaac ggagatccaa ataacatgga caaagcagtt





11761
aaactgtata ggaagcttaa gagggagata acattccatg gggccaaaga aatagcactc





11821
agttattctg ctggtgcact tgccagttgt atgggcctca tatacaacag gatgggggct





11881
gtgaccactg aagtggcatt tggcctggta tgcgcaacct gtgaacagat tgctgactcc





11941
cagcatcggt ctcataggca aatggtgaca acaaccaatc cactaatcag acatgagaac





12001
agaatggttc tagccagcac tacagctaag gctatggagc aaatggctgg atcgagtgag





12061
caagcagcag aggccatgga tattgctagt caggccaggc aaatggtgca ggcgatgaga





12121
accgttggga ctcatcctag ctccagtgct ggtctaaaag atgatcttct tgaaaatttg





12181
caggcctatc agaaacgaat gggggtgcag atgcaacgat tcaagtgatc ctctcgtcat





12241
tgcagcaaat atcattggaa tcttgcactt gatattgtgg attcttgatc gtcttttttt





12301
caaatgcatt tatcgtcgct ttaaatacgg tttgaaaaga gggccttcta cggaaggagt





12361
gccagagtct atgagggaag aatatcgaaa ggaacagcag aatgctgtgg atgttgacga





12421
tggtcatttt gtcaacatag agctggagta aaaaactacc ttgtttctac tacagacgaa





12481
catataaggc atccgaaaaa aacgttctag tcccataggc gccgactacc ggcagcggct





12541
ccgacggcag ccgaggttta cctcgacgta actggaggta caaaattaca gcgacgcctc





12601
tggcagctcc ggagctgtag cgcccccccc cacagccaga gcggccaaga caatccgaaa





12661
cggggtagac ctggacgcgg atcgcaagcc gccccggcag cgacctctag ccgccgccgc





12721
ggagagcgcg agacggtagc acccgggtag accgttccgc cgtttccgag acgccccggc





12781
agcgacccct agccgccgcc gccgcggaga gaccgagccg gacggtgccc gccgggacca





12841
ggtagaccgt tccgccgtgc cccagccacc tccgcgaagc gaccgaggta ccgttgacat





12901
tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat





12961
atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac





13021
ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc





13081
cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg





13141
tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat





13201
tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc





13261
atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt





13321
gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac





13381
caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc





13441
ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc





13501
actgcttact ggcttatcga aattaatacg actcactata gggagaccca agctggctag





13561
cgtgtcgccc ggagtactgg tcgacctccg aagttggggg ggagcgaaag caggtactga





13621
ttcaaaatgg aagattttgt gcgacaatgc ttcaatccga tgattgtcga gcttgcggaa





13681
aaggcaatga aagagtatgg agaggacctg aaaatcgaaa caaacaaatt tgcagcaata





13741
tgcactcact tggaagtgtg cttcatgtat tcagattttc acttcatcga tgagcaaggc





13801
gagtcaatag tcgtagaact tggcgatcca aatgcacttt tgaagcacag atttgaaata





13861
atcgagggaa gagatcgcac aatagcctgg acagtaataa acagtatttg caacactaca





13921
ggggctgaga aaccaaagtt tctaccagat ttgtatgatt acaagaagaa tagattcatc





13981
gaaattggag taacaaggag agaagttcac atatactatc tggaaaaggc caataaaatt





14041
aaatctgaga agacacacat ccacattttc tcattcactg gggaggaaat ggccacaaag





14101
gccgactaca ctctcgatga agaaagcagg gctaggatca aaaccaggct attcaccata





14161
agacaagaaa tggctagcag aggcctctgg gattcctttc gtcagtccga gagaggcgaa





14221
gagacaattg aagaaagatt tgaaatcaca ggaacaatgc gcaagcttgc cgaccaaagt





14281
ctcccgccaa acttctccag ccttgaaaaa tttagagcct atgtggatgg attcgaaccg





14341
aacggctaca ttgagggcaa gctttctcaa atgtccaaag aagtaaatgc tagaattgaa





14401
ccttttttga aatcaacacc acgaccactt agacttccgg atgggcctcc ctgttctcag





14461
cggtccaaat tcctgctgat ggatgcctta aaattaagca ttgaggaccc aagtcatgag





14521
ggagagggga taccgctata tgatgcaatc aaatgcatga gaacattctt tggatggaag





14581
gaacccaatg ttgttaaacc acacgaaaag ggaataaatc caaattatct tctgtcatgg





14641
aagcaagtac tggcagaact gcaggacatt gagaatgagg agaaaattcc aaggactaaa





14701
aatatgaaga aaacgagtca gttaaagtgg gcacttggtg agaacatggc accagaaaag





14761
gtagactttg acgattgtaa agatgtaggc gatttgaagc aatatgatag tgatgaacca





14821
gaattgaggt cgcttgcaag ttggattcag aatgagttca acaaggcatg tgaactgacc





14881
gattcaagct ggatagagct cgatgagatt ggagaagatg cggctccaat tgaacacatt





14941
gcaagcatga gaaggaatta tttcacagca gaggtgtctc attgcagagc cacagaatac





15001
ataatgaagg gggtgtacat caatactgcc ttgcttaatg catcctgtgc agcaatggat





15061
gatttccaat taattccaat gataagcaag tgtagaacta aggagggaag gcgaaagacc





15121
aatttgtacg gtttcatcat aaaaggaaga tcccacttaa ggaatgacac cgatgtggta





15181
aactttgtga gcatggagtt ttccctcact gacccaagac ttgaaccaca caaatgggag





15241
aagtactgtg ttcttgaggt aggagatatg cttctaagaa gtgccatagg ccatgtgtca





15301
aggcctatgt tcttgtatgt gaggacaaat ggaacctcaa aaattaaaat gaaatggggg





15361
atggaaatga ggcgttgcct ccttcagtca cttcaacaaa tcgagagtat gattgaagct





15421
gagtcctctg tcaaggagaa agacatgacc aaagagttct ttgaaaacaa atcagaaaca





15481
tggcccgttg gagagtcccc caaaggagtg gaggaaggtt ccattgggaa ggtctgcaga





15541
actttattgg caaagtcggt attcaacagc ttgtatgcat ctccacaact agaaggattt





15601
tcagctgaat caagaaaact gcttcttatc gttcaggctc ttagggacaa cctggaacct





15661
gggacctttg atcttggggg gctatatgaa gcaattgagg agtgcctgat taatgatccc





15721
tgggttttgc ttaatgcttc ttggttcaac tccttcctca cacatgcatt gagatagttg





15781
tggcaatgct actatttgct atccatactg tccaaaaaag taccttgttt ctactacaga





15841
cgaacatata aggcatccga aaaaaacgtt ctagtcccat aggcgccgac taccggcagc





15901
ggctccgacg gcagccgagg tttacctcga cgtaactgga ggtacaaaat tacagcgacg





15961
cctctggcag ctccggagct gtagcgcccc cccccacagc cagagcggcc aagacaatcc





16021
gaaacggggt agacctggac gcggatcgca agccgccccg gcagcgacct ctagccgccg





16081
ccgcggagag cgcgagacgg tagcacccgg gtagaccgtt ccgccgtttc cgagacgccc





16141
cggcagcgac ccctagccgc cgccgccgcg gagagaccga gccggacggt gcccgccggg





16201
accaggtaga ccgttccgcc gtgccccagc cacctccgcg aagcgaccga aagggcgaat





16261
tctgcagaaa gcttaagttt aaaccgctga tcagcctcga ctgtgccttc tagttgccag





16321
ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact





16381
gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt





16441
ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat





16501
gctggggatg cggtgggctc tatggttaat taagtgtcgc ccggagtact ggtcgacctc





16561
cgaagttggg ggggagcgaa agcaggagtt taaatgaatc caaaccagaa aataataacc





16621
attgggtcaa tctgtatggt agtcggaata attagcctaa tattgcaaat aggaaatata





16681
atctcaatat ggattagcca ttcaattcaa accggaaatc aaaaccatac tggaatatgc





16741
aaccaaggca gcattaccta taaagttgtt gctgggcagg actcaacttc agtgatatta





16801
accggcaatt catctctttg tcccatccgt gggtgggcta tacacagcaa agacaatggc





16861
ataagaattg gttccaaagg agacgttttt gtcataagag agccttttat ttcatgttct





16921
cacttggaat gcaggacctt ttttctgact caaggcgcct tactgaatga caagcattca





16981
agggggacct ttaaggacag aagcccttat agggccttaa tgagctgccc tgtcggtgaa





17041
gctccgtccc cgtacaattc aaggtttgaa tcggttgctt ggtcagcaag tgcatgtcat





17101
gatggaatgg gctggctaac aatcggaatt tctggtccag atgatggagc agtggctgta





17161
ttaaaataca accgcataat aactgaaacc ataaaaagtt ggaggaagaa tatattgaga





17221
acacaagagt ctgaatgtac ctgtgtaaat ggttcatgtt ttaccataat gaccgatggc





17281
ccaagtgatg ggctggcctc gtacaaaatt ttcaagatcg agaaggggaa ggttactaaa





17341
tcgatagagt tgaatgcacc taattctcac tacgaggaat gttcctgtta ccctgatacc





17401
ggcaaagtga tgtgtgtgtg cagagacaat tggcacggtt cgaaccgacc atgggtgtcc





17461
ttcgaccaaa acctagatta taaaatagga tacatctgca gtggggtttt cggtgacaac





17521
ccgcgtccca aagatggaac aggcagctgt ggcccagtgt ctgctgatgg agcaaacgga





17581
gtaaagggat tttcatataa gtatggcaat ggtgtttgga taggaaggac taaaagtgac





17641
agttccagac atgggtttga gatgatttgg gatcctaatg gatggacaga gactgatagt





17701
aggttctcta tgagacaaga tgttgtggca ataactaatc ggtcagggta cagcggaagt





17761
ttcgttcaac atcctgagct aacagggcta gactgtatga ggccttgctt ctgggttgaa





17821
ttaatcaggg ggctacctga ggaggacgca atctggacta gtgggagcat catttctttt





17881
tgtggtgtga atagtgatac tgtagattgg tcttggccag acggtgctga gttgccgttc





17941
accattgaca agtagtttgt tcaaaaaact ccttgtttct actacagacg aacatataag





18001
gcatccgaaa aaaacgttct agtcccatag gcgccgacta ccggcagcgg ctccgacggc





18061
agccgaggtt tacctcgacg taactggagg tacaaaatta cagcgacgcc tctggcagct





18121
ccggagctgt agcgcccccc cccacagcca gagcggccaa gacaatccga aacggggtag





18181
acctggacgc ggatcgcaag ccgccccggc agcgacctct agccgccgcc gcggagagcg





18241
cgagacggta gcacccgggt agaccgttcc gccgtttccg agacgccccg gcagcgaccc





18301
ctagccgccg ccgccgcgga gagaccgagc cggacggtgc ccgccgggac caggtagacc





18361
gttccgccgt gccccagcca cctccgcgaa gcgaccgagg gcccgttgac attgattatt





18421
gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt





18481
ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc





18541
attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg





18601
tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat





18661
gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca





18721
gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat





18781
taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg





18841
gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca





18901
acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg





18961
tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgctta





19021
ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct agcgtgtcgc





19081
ccggagtact ggtcgacctc cgaagttggg ggggagcaaa agcagggtag ataatcactc





19141
acagagtgac atcgaaatca tggcgaccaa aggcaccaaa cgatcttacg aacagatgga





19201
gactgatgga gaacgccaga atgccactga aatcagagca tctgtcggaa aaatgattga





19261
tggaattgga cgattctaca tccaaatgtg caccgaactt aaactcagtg attatgaggg





19321
acggctgatt cagaacagct taacaataga gagaatggtg ctctctgctt ttgacgagag





19381
gaggaataaa tatctagaag aacatcccag tgcggggaaa gatcctaaga aaactggagg





19441
acctatatac aggagagtag atggaaagtg gaggagagaa ctcatccttt atgacaaaga





19501
agaaataaga cgaatctggc gccaagctaa taatggtgac gatgcaacgg ctggtctgac





19561
tcacatgatg atctggcact ccaatttgaa tgatgcaact taccagagga caagagctct





19621
tgttcgcaca ggaatggatc ccaggatgtg ctcactgatg cagggttcaa ccctccctag





19681
gaggtctggg gccgcaggtg ctgcagtcaa aggagttgga acaatggtga tggaattgat





19741
cagaatgatc aaacgtggga tcaatgatcg gaacttctgg aggggtgaga atggacggag





19801
aacaaggatt gcttatgaaa gaatgtgcaa cattctcaaa gggaaatttc aaacagctgc





19861
acaaagaaca atggtggatc aagtgagaga gagccggaat ccaggaaatg ctgagttcga





19921
agatctcatc tttttagcac ggtctgcact catattgaga gggtcagttg ctcacaagtc





19981
ctgcctgcct gcctgtgtgt atggatctgc cgtagccagt ggatacgact ttgaaagaga





20041
gggatactct ctagtcggaa tagacccttt cagactgctt caaaacagcc aagtatacag





20101
cctaatcaga ccaaatgaga atccagcaca caagagtcaa ctggtgtgga tggcatgcca





20161
ttctgctgca tttgaagatc taagagtatc aagcttcatc agagggacga aagtggtccc





20221
aagagggaag ctttccacta gaggagttca aattgcttcc aatgaaaaca tggagactat





20281
ggaatcaagt acccttgaac tgagaagcag atactgggcc ataaggacca gaagtggagg





20341
gaacaccaat caacagaggg cttcctcggg ccaaatcagc atacaaccta cgttctcagt





20401
acagagaaat ctcccttttg acagaccaac cattatggca gcattcactg ggaatacaga





20461
ggggagaaca tctgacatga gaaccgaaat cataaggctg atggaaagtg caagaccaga





20521
agatgtgtct ttccaggggc ggggagtctt cgagctctcg gacgaaaagg caacgagccc





20581
gatcgtgccc tcctttgaca tgagtaatga aggatcttat ttcttcggag acaatgcaga





20641
ggagtacgac aattaaagaa aaataccctt gtttctacta cagacgaaca tataaggcat





20701
ccgaaaaaaa cgttctagtc ccataggcgc cgactaccgg cagcggctcc gacggcagcc





20761
gaggtttacc tcgacgtaac tggaggtaca aaattacagc gacgcctctg gcagctccgg





20821
agctgtagcg ccccccccca cagccagagc ggccaagaca atccgaaacg gggtagacct





20881
ggacgcggat cgcaagccgc cccggcagcg acctctagcc gccgccgcgg agagcgcgag





20941
acggtagcac ccgggtagac cgttccgccg tttccgagac gccccggcag cgacccctag





21001
ccgccgccgc cgcggagaga ccgagccgga cggtgcccgc cgggaccagg tagaccgttc





21061
cgccgtgccc cagccacctc cgcgaagcga ccgaaagggc gaattctgca gaaagcttaa





21121
gtttaaaccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc





21181
cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa





21241
atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg





21301
ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg





21361
gctctatggc acttacacta acacgtggtg tcgcccggag tactggtcga cctccgaagt





21421
tgggggggag caaaagcagg ggaaaataaa aacaaccaaa atgaaggcaa aactactggt





21481
cctgttatat gcatttgtag ctacagatgc agacacaata tgtataggct accatgcgaa





21541
caactcaacc gacactgttg acacaatact cgagaagaat gtggcagtga cacattctgt





21601
taacctgctc gaagacagcc acaacgggaa actatgtaaa ttaaaaggaa tagccccact





21661
acaattgggg aaatgtaaca tcaccggatg gctcttggga aatccagaat gcgactcact





21721
gcttccagcg agatcatggt cctacattgt agaaacacca aactctgaga atggagcatg





21781
ttatccagga gatctcatcg actatgagga actgagggag caattgagct cagtatcatc





21841
attagaaaga ttcgaaatat ttcccaagga aagttcatgg cccaaccaca cattcaacgg





21901
agtaacagta tcatgctccc ataggggaaa aagcagtttt tacagaaatt tgctatggct





21961
gacgaagaag ggggattcat acccaaagct gaccaattcc tatgtgaaca ataaagggaa





22021
agaagtcctt gtactatggg gtgttcatca cccgtctagc agtgatgagc aacagagtct





22081
ctatagtaat ggaaatgctt atgtctctgt agcgtcttca aattataaca ggagattcac





22141
cccggaaata gctgcaaggc ccaaagtaag agatcaacat gggaggatga actattactg





22201
gaccttgcta gaacccggag acacaataat atttgaggca actggtaatc taatagcacc





22261
atggtatgct ttcgcactga gtagagggtt tgagtccggc atcatcacct caaacgcgtc





22321
aatgcatgag tgtaacacga agtgtcaaac accccaggga gctataaaca gcaatctccc





22381
tttccagaat atacacccag tcacaatagg agagtgccca aaatatgtca ggagtaccaa





22441
attgaggatg gttacaggac taagaaacat cccatccatt caatacagag gtctatttgg





22501
agccattgct ggttttattg aggggggatg gactggaatg atagatggat ggtatggtta





22561
tcatcatcag aatgaacagg gatcaggcta tgcagcggat caaaaaagca cacaaaatgc





22621
cattaacggg attacaaaca aggtgaactc tgttatcgag aaaatgaaca ctcaattcac





22681
agctgtgggt aaagaattca acaacttaga aaaaaggatg gaaaatttaa ataaaaaagt





22741
tgatgatggg tttctggaca tttggacata taatgcagaa ttgttagttc tactggaaaa





22801
tgaaaggact ttggatttcc atgacttaaa tgtgaagaat ctgtacgaga aagtaaaaag





22861
ccaattaaag aataatgcca aagaaatcgg aaatgggtgt tttgagttct accacaagtg





22921
tgacaatgaa tgcatggaaa gtgtaagaaa tgggacttat gattatccaa aatattcaga





22981
agaatcaaag ttgaacaggg aaaagataga tggagtgaaa ttggaatcaa tgggggtgta





23041
tcagattctg gcgatctact caactgtcgc cagttcactg gtgcttttgg tctccctggg





23101
ggcaatcagt ttctggatgt gttctaatgg gtctttgcag tgcagaatat gcatctgaga





23161
ttaggatttc agaaatataa ggaaaaacac ccttgtttct actacagacg aacatataag





23221
gcatccgaaa aaaacgttct agtcccatag gcgccgacta ccggcagcgg ctccgacggc





23281
agccgaggtt tacctcgacg taactggagg tacaaaatta cagcgacgcc tctggcagct





23341
ccggagctgt agcgcccccc cccacagcca gagcggccaa gacaatccga aacggggtag





23401
acctggacgc ggatcgcaag ccgccccggc agcgacctct agccgccgcc gcggagagcg





23461
cgagacggta gcacccgggt agaccgttcc gccgtttccg agacgccccg gcagcgaccc





23521
ctagccgccg ccgccgcgga gagaccgagc cggacggtgc ccgccgggac caggtagacc





23581
gttccgccgt gccccagcca cctccgcgaa gcgaccga





Claims
  • 1. An expression vector for the production of a virus with a segmented genome comprising: (a) at least one transcription cassette for vRNA production comprising a Pol I promoter operably linked to a first cDNA from the virus linked to a Pol I transcription termination sequence; and(b) at least one transcription cassette for vRNA and mRNA production comprising a Pol I promoter operably linked to a second cDNA from the virus linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the second cDNA and a Pol II transcription termination sequence.
  • 2. The expression vector of claim 1, wherein the virus is selected from the group consisting of positive-sense RNA viruses, negative-sense RNA viruses and double-stranded RNA viruses.
  • 3. The expression vector of claim 1, wherein the vector is selected from the group consisting of a viral vector, a cosmid, phasmid, and a plasmid.
  • 4. The expression vector of claim 1, wherein the number of distinct transcription cassettes is six or more.
  • 5. The expression vector of claim 1, wherein one or multiple nuclear targeting sequences are present in an expression vector.
  • 6. The expression vector of claim 1, wherein the vector is selected from the group consisting of a low copy plasmid, and an intermediate copy plasmid.
  • 7. An expression vector capable of generating influenza virus, the expression vector comprising: (a) four transcription cassettes for vRNA production, the transcription cassettes for vRNA production comprising a Pol I promoter operably linked to an influenza virus HA cDNA linked to a Pol I transcription termination sequence; a Pol I promoter operably linked to an influenza virus NA cDNA linked to a Pol I transcription termination sequence; a Pol I promoter operably linked to an influenza virus M cDNA linked to a Pol I transcription termination sequence; and a Pol I promoter operably linked to an influenza virus NS cDNA linked to a Pol I transcription termination sequence; and(b) four transcription cassettes that each produce vRNA and mRNA, the transcription cassettes comprising a Pol I promoter operably linked to an influenza virus PA cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PA cDNA and a Pol II transcription termination sequence; a Pol I promoter operably linked to an influenza virus PB1 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB1 cDNA and a Pol II transcription termination sequence; a Pol I promoter operably linked to an influenza virus PB2 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB2 cDNA and a Pol II transcription termination sequence; and a Pol I promoter operably linked to an influenza virus NP cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the NP cDNA and a Pol II transcription termination sequence.
  • 8. The expression vector of claim 7, wherein the vector is selected from the group consisting of a viral vector, a cosmid, phasmid, and a plasmid.
  • 9. The expression vector of claim 7, wherein the vector is selected from the group consisting of a low copy plasmid, and an intermediate copy plasmid.
  • 10. The expression vector of claim 7, wherein one vector comprises one or multiple nuclear targeting sequences.
  • 11. The expression vector of claim 7, wherein the transcription cassettes are arranged in the expression vector in pairs of vRNA transcription cassettes and vRNA and mRNA transcription cassettes.
  • 12. The expression vector of claim 7, wherein the influenza virus is influenza A virus.
  • 13. The expression vector of claim 7, wherein the expression vector is selected from the group consisting of pYA4519 and pYA4562.
  • 14. An expression vector comprising: (a) at least one transcription cassette for vRNA production, the transcription cassette selected from the group consisting of a Pol I promoter operably linked to an influenza virus HA cDNA linked to a Pol I transcription termination sequence; a Pol I promoter operably linked to an influenza virus NA cDNA linked to a Pol I transcription termination sequence; a Pol I promoter operably linked to an influenza virus M cDNA linked to a Pol I transcription termination sequence; and a Pol I promoter operably linked to an influenza virus NS cDNA linked to a Pol I transcription termination sequence; and(b) four transcription cassettes that each produce vRNA and mRNA, the transcription cassettes comprising a Pol I promoter operably linked to an influenza virus PA cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PA cDNA and a Pol II transcription termination sequence; a Pol I promoter operably linked to an influenza virus PB1 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB1 cDNA and a Pol II transcription termination sequence; a Pol I promoter operably linked to an influenza virus PB2 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB2 cDNA and a Pol II transcription termination sequence; and a Pol I promoter operably linked to an influenza virus NP cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the NP cDNA and a Pol II transcription termination sequence.
  • 15. The expression vector of claim 14, wherein the vector comprises two transcription cassettes for vRNA production, the transcription cassettes comprising a Pol I promoter operably linked to an influenza virus M cDNA linked to a Pol I transcription termination sequence; and a Pol I promoter operably linked to an influenza virus NS cDNA linked to a Pol I transcription termination sequence.
  • 16. The expression vector of claim 14, further comprising a second expression vector for the production HA cDNA, and NA cDNA.
  • 17. The expression vector of claim 16, further comprising a second expression vector for the production of HA cDNA, and a third expression vector for the production of NA cDNA.
  • 18. The expression vector of claim 14, wherein the vector is selected from the group consisting of a viral vector, a cosmid, phasmid, and a plasmid.
  • 19. The expression vector of claim 14, wherein the expression vector further comprises one or multiple nuclear targeting sequences.
  • 20. The expression vector of claim 14, wherein the influenza virus is influenza A virus.
  • 21. A method for the production of influenza virus in a eukaryotic cell, the method comprising introducing an expression vector into the eukaryotic cell, the expression vector comprising: (a) four transcription cassettes for vRNA production, the transcription cassettes for vRNA production comprising a Pol I promoter operably linked to an influenza virus HA cDNA linked to a Pol I transcription termination sequence; a Pol I promoter operably linked to an influenza virus NA cDNA linked to a Pol I transcription termination sequence; a Pol I promoter operably linked to an influenza virus M cDNA linked to a Pol I transcription termination sequence; and a Pol I promoter operably linked to an influenza virus NS cDNA linked to a Pol I transcription termination sequence; and(b) four transcription cassettes that each produce vRNA and mRNA, the transcription cassettes comprising a Pol I promoter operably linked to an influenza virus PA cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PA cDNA and a Pol II transcription termination sequence; a Pol I promoter operably linked to an influenza virus PB1 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB1 cDNA and a Pol II transcription termination sequence; a Pol I promoter operably linked to an influenza virus PB2 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB2 cDNA and a Pol II transcription termination sequence; and a Pol I promoter operably linked to an influenza virus NP cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the NP cDNA and a Pol II transcription termination sequence.
  • 22. A method for the production of influenza virus in a eukaryotic cell, the method comprising introducing an expression vector into the eukaryotic cell, the expression vector comprising: (a) two transcription cassettes for vRNA production, the transcription cassettes for vRNA production comprising a Pol I promoter operably linked to an influenza virus M cDNA linked to a Pol I transcription termination sequence; and a Pol I promoter operably linked to an influenza virus NS cDNA linked to a Pol I transcription termination sequence; and(b) four transcription cassettes that each produce vRNA and mRNA, the transcription cassettes comprising a Pol I promoter operably linked to an influenza virus PA cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PA cDNA and a Pol II transcription termination sequence; a Pol I promoter operably linked to an influenza virus PB1 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB1 cDNA and a Pol II transcription termination sequence; a Pol I promoter operably linked to an influenza virus PB2 cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the PB2 cDNA and a Pol II transcription termination sequence; and a Pol I promoter operably linked to an influenza virus NP cDNA linked to a Pol I transcription termination sequence and a Pol II promoter operably linked to the NP cDNA and a Pol II transcription termination sequence.
GOVERNMENTAL RIGHTS

This invention was made with government support under RO1 AI065779 awarded by the National Institutes of Health. The government has certain rights in the invention.

Provisional Applications (1)
Number Date Country
61168996 Apr 2009 US